Team:Kyoto/Engineering
Engineering Success
The following is a record of our Engineering Flow.
We have set a major goal to make the plant production more sustainable.
To achieve this goal, we established the four measures.
For each topic, we proceeded with the project in accordance with the Principles of Engineering.
To achieve this goal, we established the four measures.
For each topic, we proceeded with the project in accordance with the Principles of Engineering.
Click each title to see detail
1. Virus and Viroid detection
2. Feeding RNAi for western flower thrips
3. Other applications for cut flower maintenance
4. Biomolecular Production Platform
Virus and Viroid detection
Cycle1
We performed CRISPR-Cas12a mediated LAMP assay to detect DNA viruses including Dahlia Mosaic Virus (DMV) using commercially available enzymes Cycle2
We performed CRISPR-Cas12a mediated RT-LAMP assay to detect RNA viruses such as CSVd or TSWV using purchased enzymes Cycle3
We reduced the cost of one reaction by producing homemade enzymes Cycle4
We created hardware which helps farmers to do point-of-care testing Cycle5
We created image recognition software to improve the performance of the CRISPR-Cas12a-mediated LAMP/RT-LAMP assay Cycle6
We designed a portable detection package and improved a cycle of diagnostic imaging accuracy
We performed CRISPR-Cas12a mediated LAMP assay to detect DNA viruses including Dahlia Mosaic Virus (DMV) using commercially available enzymes Cycle2
We performed CRISPR-Cas12a mediated RT-LAMP assay to detect RNA viruses such as CSVd or TSWV using purchased enzymes Cycle3
We reduced the cost of one reaction by producing homemade enzymes Cycle4
We created hardware which helps farmers to do point-of-care testing Cycle5
We created image recognition software to improve the performance of the CRISPR-Cas12a-mediated LAMP/RT-LAMP assay Cycle6
We designed a portable detection package and improved a cycle of diagnostic imaging accuracy
Cycle1
We performed CRISPR-Cas12a mediated LAMP assay to detect DNA viruses including Dahlia Mosaic Virus (DMV) using commercially available enzymes
We performed CRISPR-Cas12a mediated LAMP assay to detect DNA viruses including Dahlia Mosaic Virus (DMV) using commercially available enzymes
Design
We first tried to detect Dahlia Mosaic Virus (DMV) which has become a serious problem for the Japanese
flower industry. When dahlias are infected with the virus, their leaves show weird patterns, losing
their commercial value and ending up being discarded. When we interviewed several experts during human
practice, we learned that RT-qPCR is now commonly used by them to detect the virus in dahlia bulbs.
However, since the RT-qPCR method requires expensive and complicated equipment and knowledge of
molecular biology, farmers are not able to detect virus infections by themselves. To solve this
problem, we worked to create a new detection method for DMV that is easy to handle[1.1].Therefore, we
focused on the LAMP method (Loop-Mediated Isothermal Amplification)[1.2].
The LAMP method amplifies
DNAs
simply by incubating reactions at isothermal temperatures. Since it is easier and faster than PCR, it
is frequently used for virus detection.[1.3][1.4]
How does the LAMP method work?
In the LAMP method, a strand-displacing B. stearothermophilus DNA polymerase initiates DNA synthesis from primers attached to the end of the target sequence. The ssDNA amplicon forms a loop at the end of it, which works as a new template for further DNA amplification. Thus, DNA is amplified exponentially.[1.5]
However, one of the disadvantages of this LAMP method is that it is likely to produce many pseudo-positive samples due to non-specific amplification. This is a fatal problem because the unnecessary disposal of pseudo-positive lines also leads to economic losses for the flower industry. To compensate for this weakness of the LAMP method, we decided to combine the LAMP method and CRISPR-Cas12a fluorescence assay.[1.6]
How does the CRISPR-Cas12a fluorescence assay work?
When CRISPR-Cas12a binds to crRNA, they form an RNP complex (ribonucleoprotein complex). When the RNP complex recognizes dsDNA that has complementary base-pairing to the guide crRNA, it acquires non-specific ssDNA cleavage activity, collateral activity. If a probe composed of a fluorophore (FAM) and a quencher connected by a 5-nucleotide sequence (TTATT) was cleaved by the activated Cas12a, it displays increased fluorescence[1.7]. This CRISPR-Cas12a fluorescence assay enhances specificity for nucleic acid detection significantly[1.8].
How does the LAMP method work?
In the LAMP method, a strand-displacing B. stearothermophilus DNA polymerase initiates DNA synthesis from primers attached to the end of the target sequence. The ssDNA amplicon forms a loop at the end of it, which works as a new template for further DNA amplification. Thus, DNA is amplified exponentially.[1.5]
However, one of the disadvantages of this LAMP method is that it is likely to produce many pseudo-positive samples due to non-specific amplification. This is a fatal problem because the unnecessary disposal of pseudo-positive lines also leads to economic losses for the flower industry. To compensate for this weakness of the LAMP method, we decided to combine the LAMP method and CRISPR-Cas12a fluorescence assay.[1.6]
How does the CRISPR-Cas12a fluorescence assay work?
When CRISPR-Cas12a binds to crRNA, they form an RNP complex (ribonucleoprotein complex). When the RNP complex recognizes dsDNA that has complementary base-pairing to the guide crRNA, it acquires non-specific ssDNA cleavage activity, collateral activity. If a probe composed of a fluorophore (FAM) and a quencher connected by a 5-nucleotide sequence (TTATT) was cleaved by the activated Cas12a, it displays increased fluorescence[1.7]. This CRISPR-Cas12a fluorescence assay enhances specificity for nucleic acid detection significantly[1.8].
Build
We designed primers for the LAMP method of DMV[1.9].
In the design of crRNAs for CRISPR-Cas12a, the suitable sites for crRNAs were determined in the sequences to be amplified by the RT-LAMP method[1.10].
In the design of crRNAs for CRISPR-Cas12a, the suitable sites for crRNAs were determined in the sequences to be amplified by the RT-LAMP method[1.10].
Test
We performed CRISPR-Cas12a-mediated LAMP assay[1.11] against DMV using a
purchased B.
stearothermophilus
DNA polymerase, Bst3.0 (New England BIolabs). We made a series of dilution of DMV target DNA
(synthesized by IDT) from 1010 copies to 1 copy for the LAMP reaction to
verify the detection
limit. From the value of FAM, we confirmed that the detection limit is 1 copy.
Learn
Since we succeeded in CRISPR-Cas12a-mediated LAMP assay for DMV that is a DNA virus, we decided to
proceed to CRISPR-Cas12a-mediated RT-LAMP assay to detect other RNA viruses.
Cycle2
We performed CRISPR-Cas12a mediated RT-LAMP assay to detect RNA viruses such as CSVd or TSWV using purchased enzymes
We performed CRISPR-Cas12a mediated RT-LAMP assay to detect RNA viruses such as CSVd or TSWV using purchased enzymes
Design
We reverse-transcribed RNA into cDNA, and then tried to detect RNA viruses such as CSVd and TSWV in
the same way as CRISPR-Cas12a mediated LAMP assay.
Build
We designed primers for RT-LAMP assay against CSVd and TSWV[1.12][1.13].
In the design of crRNAs for CRISPR-Cas12a, the suitable sites for crRNAs were determined in the sequences to be amplified by the RT-LAMP method.
In the design of crRNAs for CRISPR-Cas12a, the suitable sites for crRNAs were determined in the sequences to be amplified by the RT-LAMP method.
Test
In this experiment, we aimed to detect CSVd and TSWV using RNA samples obtained from actual infected
plants. We got these RNA samples from a collaborator. He kindly gave us two RNA samples of each.
Since BST polymerase 2.0 has reverse transcription activity, we used twice the amount of the enzyme to perform the RT-LAMP reaction. Since we were told to dilute the samples 20 times, we prepared a 3-step gradient of samples: original concentration, diluted 20 times, and a no-template one. For RT-LAMP reaction, samples in the following table were incubated at 63°C for 30 minutes.
Since BST polymerase 2.0 has reverse transcription activity, we used twice the amount of the enzyme to perform the RT-LAMP reaction. Since we were told to dilute the samples 20 times, we prepared a 3-step gradient of samples: original concentration, diluted 20 times, and a no-template one. For RT-LAMP reaction, samples in the following table were incubated at 63°C for 30 minutes.
After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also measured the value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient
fluorescence could not be confirmed.
We also performed Cas12a-mediated RT-LAMP assay on TSWV samples. For TSWV we prepared two types of
crRNA, and we performed the RT-LAMP reaction by incubating the samples in the following table at 63°C
for 30 min.
After the assay, we checked the fluorescence of FAM with the naked eye under a blue light reader. We
also
measured the value of FAM quantitatively by a qPCR machine, but as shown in the graph, sufficient
fluorescence could not be confirmed this time ,either. The problems seem to be that the quality of the
RNA
samples was not confirmed and the optimal conditions for BST2.0 warm start were not sufficiently
examined,
so we would like to solve these in the future.
Learn
We found through Cycle1 and Cycle2 that a purchased BST DNA polymerase can be successfully used for
virus or viroid detection. However, the assay using purchased BST 3.0 / BST2.0 warmstart in the
recommended protocol would cost about $0.5 per reaction. Considering that the typical market price of
dahlia is about $7, it would be difficult for farmers to test all the crops and completely eliminate
the
spread of the viruses. Thus, in order to reduce the cost, we decided to produce enzymes by ourselves.
Cycle3
We reduced the cost of one reaction by producing homemade enzymes
We reduced the cost of one reaction by producing homemade enzymes
Design
We considered producing the patent expired enzymes (BST DNA polymerase, Reverse Transcriptase) by
ourselves to lower the price per one reaction.
Build
We decided to prepare HIV-1 reverse transcriptase and B. stearothermophilus DNA polymerase. For HIV-1
RT, a peptide made from one ORF is partially digested in an infected cell and becomes two fragments,
p66
and p51. It is known that the HIV-1 RT functions when the p66 and p51 form a heterodimer. To purify
them
as recombinant proteins in E.coli, we separately expressed and purified p66 and p51, and dimerized
them
in vitro before use[1.14][1.15].
Test
We performed the purification of proteins in the following steps.
In addition to our homemade DNA polymerase, we examined our homemade reverse transcriptase activity in
vitro. We used total yeast DNA/RNA for this assay. We selected the intron-containing region of Rpl19A
as
a template for reverse transcription. If there is no reverse transcription, the genomic DNA including
an
intron is amplified by PCR, resulting in a band of 876 bp. On the other hand, if cDNA is correctly
synthesized by reverse transcription, a band of 370 bp without an intron should appear.
As shown below, the cDNA band was successfully observed in an RT-dependent manner. From this result, we concluded the activity of homemade RT[1.17].
(From left to right: 1-4: newly prepared homemade RT with different enzyme concentrations, 5: no
enzyme,
6: BST3.0, 7: old homemade RT, 8: no template control)
- Transformation
The genes of interest were cloned into pET-11a and were transformed into BL21 (DE3). A lot of colonies were observed in all the plates. - Protein expression
Each LB culture was grown at 37 °C to the log-phase. When OD600 reaches 0.35-0.7, we added IPTG at the final concentration of 0.5 mM. After 3 hours of induction, cells were harvested and frozen in liquid-N2. - Protein purification
After the cell disruption by sonication, each protein was purified using Ni-NTA beads. Samples were eluted by a four-step gradient of Imidazole. We observed the correct bands in the SDS-PAGE gel (shown below). The arrowheads indicate the targeted proteins. - Dialysis
We put the purified proteins in dialysis bags and incubated them o/n at 4°C in 500 mL of storage buffer. - Assay
We conducted the LAMP reaction against DMV with the purified homemade DNA polymerase. As shown in the figure below, we observed the ladder-like pattern, which is a typical pattern of LAMP reaction[1.16]. We concluded that the LAMP reaction by homemade DNA polymerase is a success. Isothermal Buffer 1 is a condition for increasing the DNA polymerase activity of BST3.0. Isothermal Buffer 2 is for increasing the reverse transcription activity of the enzyme.
As shown below, the cDNA band was successfully observed in an RT-dependent manner. From this result, we concluded the activity of homemade RT[1.17].
Learn
We successfully produced the two enzymes necessary for RT-LAMP and confirmed their activities. We
reduced the cost of the reaction through Cycle3, which brought us one step forward to the practical
application of point-of-care detection at farms. However, there is one problem that we have to solve
for
practical application. That is, “how to measure the fluorescence generated by this assay”.
Cycle4
Create hardware that allows farmers to check detection results on site
Create hardware that allows farmers to check detection results on site
Design
To address this problem, we decided to develop hardware that anyone can use easily and quickly. We
designed hardware that enables visual judgment.
Build
We designed the hardware named “DLAMI” (diseased leaves assessment, machine interface) for
fluorometry.
DLAMI hardware is a black box equipped PCR tube stand with a lid. There are two holes in
the sides of the box. The bigger hole is used for a 470 nm blue LED for the excitation of 5-FAM
fluorescence (maximum absorption wavelength at 495 nm, maximum emission at 520 nm)[1.18]. The smaller
hole
is
a camera hole by which we acquire the sample images. In the front of the camera hole, we inserted a
525
nm long-pass filter to cut off the irradiation light.
Test
DLAMI hardware was constructed as shown below.
DLAMI was tested with a PCR tube containing virus DNA template. As clearly shown in picture below, we
could take a picture of the tube with fluorescence by a camera of our smartphone. Thus, we concluded
that DLAMI can be used as a portable dark room for fluorescence detection by a smartphone.
Learn
We interviewed Mr. Kurokawa, who is a flower farmer in Kyoto, to confirm whether DLAMI hardware is
useful for farmers as we expected. We showed him the blueprint of DLAMI hardware and RT-LAMP assay.
We
received a number of feedback from him which were used to improve our system.
Cycle5
Image diagnosis as a pre-screening system
Image diagnosis as a pre-screening system
Design
So far we succeeded in creating a detection system that farmers can use on site. To improve this
system, we also developed an image diagnosis software to increase the probability of a positive
result
as a pre-screening. For tests involving nucleic acid amplification such as LAMP and PCR, it is
important to increase the prior probability of a positive result through pre-screening to minimize
false positives. For this purpose, we have created DLAEMON (Diseased Leaves Assessment by Efficient
Machine-learning On Neural network). This is an innovative diagnostic imaging software that allows
AI
to automatically diagnose diseases, by just taking an image of a leaf and clicking the diagnose
button. By using this software prior to CRISPR-Cas12a assisted RT-LAMP test, the probability of
pre-diagnosis is greatly increased, contributing to a significant reduction in false positives. We
also developed a related software NOBITA (Numericalization Of Brightness of Image for one Touch
Assessment) for the quantification of the fluorescence after Cas12a reaction.
Build
For DLAEMON, we used leaf images downloaded from the public dataset, plant village[1.19]. Since our
enzyme
detection method targets dahlia viruses, initially we would have preferred to use dahlia leaf data.
However, it was hard to collect a large amount of dahlia leaf data. We decided to use cherry blossom
leaves instead to build and validate the algorithm. This consisted of 1146 pieces of training data,
360 pieces of validation data (health:disease=5:5), and 400 pieces of test data
(health:disease=5:5).
Fine-tuning[1.20] was performed using the pre-trained Resnet18.
Test
The accuracy of DLAEMON was examined and proved to be 99.9% for the training data, 99.7% for the
validation data, and 99.8% for the test data.
We also developed NOBITA, which automatically converts uploaded photos into black and white square
images and outputs the brightness of the clicked location. Pillow, an image processing library, is
used as the conversion tool.
Learn
Regarding to DLAEMON, the accuracy is very good for all the training, validation and testing data. It
is
expected that further fine-tuning using the model developed in this study will make it easier to
develop
models for other plant leaves. This suggests that it is likely that we will be able to increase the
prior probability of experiments for detection and provide stronger support for experimental results
for
more plants in the future. However, we realize that the images in the data set used in this study were
carefully taken one by one, and the distribution is far from that of the actual images taken by
farmers.
Therefore, we need to collect more divergent image data in order to create a model that can be applied
to the field.
Regarding to NOBITA, the comparison with ImageJ shows that luminance can be extracted with very high accuracy. However, there is room for improvement, too. It would be more user-friendly if it could automatically identify the location of the samples and quantitate those areas automatically. It would be also important to determine a specific threshold of the fluorescence intensity by further experiments.
Regarding to NOBITA, the comparison with ImageJ shows that luminance can be extracted with very high accuracy. However, there is room for improvement, too. It would be more user-friendly if it could automatically identify the location of the samples and quantitate those areas automatically. It would be also important to determine a specific threshold of the fluorescence intensity by further experiments.
Cycle6
Feed forward loop between image diagnosis and a portable device for enzymatic test
Feed forward loop between image diagnosis and a portable device for enzymatic test
Design
We built a portable detection device that integrates three components: an enzymatic method to detect
viral infection, hardware for fluorescence detection, and imaging software to increase the prior
probability. Using our system, newly acquired images of leaves are linked to the corresponding results
of (RT)-LAMP. These data sets should be in turn useful to improve DLAEMON software. As more image
information is accumulated through inspections, more practical machine learning that takes into
account
the blurring of photographic techniques in the field will become possible. Those feed forward loop
will
greatly contribute to improving the accuracy of diagnosis.
Eventually, DLAEMON software may reach an accuracy where a definitive diagnosis can be made without examination by enzymatic reactions. To achieve this, the level of accuracy will need to be carefully determined, taking into account factors such as the selling price of the flowers, the degree of decline in the value of the product due to the virus, and the speed of transmission of the virus.
As a prospect, we also plan to develop a drone equipped with this software. This drone will be hovering and taking thousands of photos of plant leaves and screening the infected ones in a short period of time. The farmer only needs to visit the suspected areas of the field to perform the CRISPR-Cas12a assisted LAMP / RT-LAMP assay with DLAMI hardware. We hope our system becomes the groundwork to establish such efficient agriculture.
Eventually, DLAEMON software may reach an accuracy where a definitive diagnosis can be made without examination by enzymatic reactions. To achieve this, the level of accuracy will need to be carefully determined, taking into account factors such as the selling price of the flowers, the degree of decline in the value of the product due to the virus, and the speed of transmission of the virus.
As a prospect, we also plan to develop a drone equipped with this software. This drone will be hovering and taking thousands of photos of plant leaves and screening the infected ones in a short period of time. The farmer only needs to visit the suspected areas of the field to perform the CRISPR-Cas12a assisted LAMP / RT-LAMP assay with DLAMI hardware. We hope our system becomes the groundwork to establish such efficient agriculture.
References
1.1 (JAPANESE) 球根増殖コンソーシアム ダリアのウイルス・ウイロイド病 診断マニュアル
(2019)
http://www.pref.miyazaki.lg.jp/sogonogyoshikenjo/shigoto/nogyo/topix/documents/44689_20190624121204-1.pdf
1.2 Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) "Loop-mediated isothermal amplification of DNA", Nucleic Acids Res. 28, E63.
1.3 (2019a). From PCR to LAMP: The evolution of rapid-testing in food safety. https://food-safety-news.3m.com/fsn/lamp-vs-pcr/
1.4 Zhang, Y., Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R.O., Wei, H., and Tanner, N.A. (2020) "Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP", (medRxiv).
1.5 New England Biolabs Loop-Mediated Isothermal Amplification
https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
1.6 Broughton, J.P., Deng, X., Yu, G., Fasching, C.L., Servellita, V., Singh, J., Miao, X., Streithorst, J.A., Granados, A., Sotomayor-Gonzalez, A., et al. (2020) "CRISPR-Cas12-based detection of SARS-CoV-2", Nat. Biotechnol. 38, 870–874.
1.7 Chen, J.S., Ma, E., Harrington, L.B., Da Costa, M., Tian, X., Palefsky, J.M., and Doudna, J.A. (2018) "CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity", Science 360, 436–439.
1.8 Nguyen, L.T., Smith, B.M., and Jain, P.K. (2020) "Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection", Nat. Commun. 11, 4906.
1.9 Iftikhar, R., Moyo, L., and Pappu, H.R. (2020) "A loop‐mediated isothermal amplification assay for the detection ofDahlia mosaic caulimovirusin Dahlia (Dahlia variabilis)", Ann. Appl. Biol. 176, 203–209.
1.10 New England Biolabs FAQ: How do I design a guide RNA for use with EnGen Lba Cas12a?
https://www.neb.com/faqs/2018/05/03/how-do-i-design-a-guide-rna-for-use-with-engen-lba-cas12a
1.11 Ali, Z., Aman, R., Mahas, A., Rao, G.S., Tehseen, M., Marsic, T., Salunke, R., Subudhi, A.K., Hala, S.M., Hamdan, S.M., et al. (2020) "iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2", Virus Res. 288, 198129.
1.12 Fukuta, S., Ohishi, K., Yoshida, K., Mizukami, Y., Ishida, A., and Kanbe, M. (2004) "Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum", J. Virol. Methods 121, 49–55.
1.13 Nagamine, K., Hase, T., and Notomi, T. (2002) "Accelerated reaction by loop-mediated isothermal amplification using loop primers", Mol. Cell. Probes 16, 223–229.
1.14 Seckler, J.M., Howard, K.J., Barkley, M.D., and Wintrode, P.L. (2009) "Solution structural dynamics of HIV-1 reverse transcriptase heterodimer", Biochemistry 48, 7646–7655.
1.15 https://biotechlink.org/1-2017/article6 (Link expired)
1.16 Lee, S.H., Baek, Y.H., Kim, Y.-H., Choi, Y.-K., Song, M.-S., and Ahn, J.-Y. (2016) "One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV", Front. Microbiol. 7, 2166.
1.17 Kellner, M.J., Ross, J.J., Schnabl, J., Dekens, M.P.S., Heinen, R., Grishkovskaya, I., Bauer, B., Stadlmann, J., Menéndez-Arias, L., Fritsche-Polanz, R., et al. (2020) "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing", bioRxiv
1.18 OPTO SCIENCE Filter finder https://www.optoscience.com/maker/omega/finder/
1.19 Ali, A. PlantVillage Dataset. https://www.kaggle.com/abdallahalidev/plantvillage-dataset
1.20 Radenovic, F., Tolias, G., and Chum, O. (2019) "Fine-Tuning CNN Image Retrieval with No Human Annotation", IEEE Trans. Pattern Anal. Mach. Intell. 41, 1655–1668.
http://www.pref.miyazaki.lg.jp/sogonogyoshikenjo/shigoto/nogyo/topix/documents/44689_20190624121204-1.pdf
1.2 Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000) "Loop-mediated isothermal amplification of DNA", Nucleic Acids Res. 28, E63.
1.3 (2019a). From PCR to LAMP: The evolution of rapid-testing in food safety. https://food-safety-news.3m.com/fsn/lamp-vs-pcr/
1.4 Zhang, Y., Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R.O., Wei, H., and Tanner, N.A. (2020) "Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP", (medRxiv).
1.5 New England Biolabs Loop-Mediated Isothermal Amplification
https://international.neb.com/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification/loop-mediated-isothermal-amplification-lamp
1.6 Broughton, J.P., Deng, X., Yu, G., Fasching, C.L., Servellita, V., Singh, J., Miao, X., Streithorst, J.A., Granados, A., Sotomayor-Gonzalez, A., et al. (2020) "CRISPR-Cas12-based detection of SARS-CoV-2", Nat. Biotechnol. 38, 870–874.
1.7 Chen, J.S., Ma, E., Harrington, L.B., Da Costa, M., Tian, X., Palefsky, J.M., and Doudna, J.A. (2018) "CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity", Science 360, 436–439.
1.8 Nguyen, L.T., Smith, B.M., and Jain, P.K. (2020) "Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection", Nat. Commun. 11, 4906.
1.9 Iftikhar, R., Moyo, L., and Pappu, H.R. (2020) "A loop‐mediated isothermal amplification assay for the detection ofDahlia mosaic caulimovirusin Dahlia (Dahlia variabilis)", Ann. Appl. Biol. 176, 203–209.
1.10 New England Biolabs FAQ: How do I design a guide RNA for use with EnGen Lba Cas12a?
https://www.neb.com/faqs/2018/05/03/how-do-i-design-a-guide-rna-for-use-with-engen-lba-cas12a
1.11 Ali, Z., Aman, R., Mahas, A., Rao, G.S., Tehseen, M., Marsic, T., Salunke, R., Subudhi, A.K., Hala, S.M., Hamdan, S.M., et al. (2020) "iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2", Virus Res. 288, 198129.
1.12 Fukuta, S., Ohishi, K., Yoshida, K., Mizukami, Y., Ishida, A., and Kanbe, M. (2004) "Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum", J. Virol. Methods 121, 49–55.
1.13 Nagamine, K., Hase, T., and Notomi, T. (2002) "Accelerated reaction by loop-mediated isothermal amplification using loop primers", Mol. Cell. Probes 16, 223–229.
1.14 Seckler, J.M., Howard, K.J., Barkley, M.D., and Wintrode, P.L. (2009) "Solution structural dynamics of HIV-1 reverse transcriptase heterodimer", Biochemistry 48, 7646–7655.
1.15 https://biotechlink.org/1-2017/article6 (Link expired)
1.16 Lee, S.H., Baek, Y.H., Kim, Y.-H., Choi, Y.-K., Song, M.-S., and Ahn, J.-Y. (2016) "One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV", Front. Microbiol. 7, 2166.
1.17 Kellner, M.J., Ross, J.J., Schnabl, J., Dekens, M.P.S., Heinen, R., Grishkovskaya, I., Bauer, B., Stadlmann, J., Menéndez-Arias, L., Fritsche-Polanz, R., et al. (2020) "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing", bioRxiv
1.18 OPTO SCIENCE Filter finder https://www.optoscience.com/maker/omega/finder/
1.19 Ali, A. PlantVillage Dataset. https://www.kaggle.com/abdallahalidev/plantvillage-dataset
1.20 Radenovic, F., Tolias, G., and Chum, O. (2019) "Fine-Tuning CNN Image Retrieval with No Human Annotation", IEEE Trans. Pattern Anal. Mach. Intell. 41, 1655–1668.
Feeding RNAi for western flower thrips
Cycle1
Preparation of in vitro transcript is expensive.
Preparation of in vitro transcript is expensive.
Design
Currently, chemical pesticides are the most commonly used method to control
Frankliniella
occidentalis damage on flowers. However, many Frankliniella occidentalis have already
become
resistant to chemical pesticides.The damage caused by these tiny insects is increasing
year
by year.
We focused on feeding RNAi to kill these insects, based on previous studies showing that
oral ingestion of dsRNA can induce RNAi-induced gene knockdown in Frankliniella
occidentalis. To this end, we selected the essential gene vATPase-B in F. occidentalis,
as a
target of RNAi[2.1].
First, we planned to produce our dsRNA by in vitro transcription using the T7 RNA
polymerase.
Test
We transcribed several dsRNAs using a commercially available kit (Megascript kit,
Thermo).
Learn
However, in vitro transcript is still too expensive to be used as a pesticide. Also, as
the
yield of the system was not so high, we were not able to obtain the sufficient amount of
dsRNA for our experiments.
Cycle2
Preparation of dsRNA in vivo
Preparation of dsRNA in vivo
Design
We decided to use in vivo dsRNA production system to obtain the sufficient amount of
dsRNA
for our experiments. To produce dsRNA in vivo, we used E. coli strain HT115 (DE3) and
plasmid vector L4440[2.2][2.3][2.4].
・HT115(DE3)
HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express T7 RNA polymerase from an IPTG-inducible promoter. This strain lacks RNase III which is a dsRNA-specific nuclease. This mutation allows the strain to produce large amounts of foreign dsRNA.
・L4440
・HT115(DE3)
HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express T7 RNA polymerase from an IPTG-inducible promoter. This strain lacks RNase III which is a dsRNA-specific nuclease. This mutation allows the strain to produce large amounts of foreign dsRNA.
・L4440
L4440 is a plasmid vector having two inverted T7 promoters with a multi-cloning site in
between. A part of the target gene is inserted into the multi-cloning site. The target
sequence is transcribed from both sides, so dsRNA can be obtained when both transcripts
anneal.
Build
We created a series of plasmids which contain a target gene sequence between two T7
promoters.
To this end, L4440 was cut with restriction enzymes and the target gene fragment was
inserted by
DNA ligation.
Test
We constructed 3 plasmids for dsRNA production. Using these plasmids, we expressed 3 dsRNAs
in
HT115(DE3) in parallel.
We tested two different protocols for the purification[2.5]. To estimate
the amounts of dsRNA
recovered in each round, purified nucleic acids were separated by 8% PAGE.
(1. vATPase-B 1, 2. vATPase-B 2, 3. EPH1, 4. L4440, 5. vATPase-B 1, 6. vATPase-B 2, 7. EPH1, 8.
L4440)
(1. L4440 (add production) (1ug), 2. vATPase-B (add production) (7ug), 3.
vATPase-B (add
production) (1ug ), 4. vATPase-B-1 (1ug), 5. vATPase-B-2 (1ug), in vitro RNA transcription (6.
vATPase-B,7. Trxz,8 EPH1 ) 1ug each)
In the first round of production, we observed a major band of vATPase-B around 700 bp, which
corresponds to the target product length. The band visible in EPH1 is slightly shorter than
that
of vATPase-B, corresponding to the shorter length of the product. In summary, we obtained a
certain amount of the target dsRNA in this preparation.
In the second round of trials, the yield of each dsRNAs was much higher than the previous
samples. This could be because the purification protocol was simplified at this time. We
noticed that a certain amount of samples could be lost during preparation.
Cycle3
Running a dsRNA uptake assay in Frankliniella occidentalis
Running a dsRNA uptake assay in Frankliniella occidentalis
Design
Using our dsRNA sample which targets vATPase-B gene of thrips, we
tested the insecticidal effect of feeding RNAi.
Previous research reported that thrips can be killed by feeding dsRNA of the
same gene as we used. However, in the paper, the authors used purified in vitro
transcripts as dsRNAs. As we prepare our dsRNA in vivo, our RNA contains a lot of host
RNA species including tRNAs and rRNAs. We examined the effect of these
contaminated materials on the feeding RNAi assay.
"RNAi" (RNA interference) is a process in which externally introduced dsRNA suppresses the
expression of genes that have complementary sequences to the dsRNA[2.6].
"vATPase-B" vATPase-B is a gene that encodes a protein that is part of a subunit of vATPase, a type of ATP-dependent proton pump that regulates the pH of various cell organelles. Since this gene is essential for thrips, knockdown of vATPase-B results in the death of the thrips.
- dsRNA is taken in from the outside.
- An enzyme called Dicer cleaves dsRNA to form a short RNA strand called siRNA.
- The siRNA forms a complex called RISC (RNA-induced silencing complex) with proteins such as Argonaute protein.
- Target genes are silenced.
"vATPase-B" vATPase-B is a gene that encodes a protein that is part of a subunit of vATPase, a type of ATP-dependent proton pump that regulates the pH of various cell organelles. Since this gene is essential for thrips, knockdown of vATPase-B results in the death of the thrips.
Build
Pipette 200 µl of 200 ng/ul dsRNA solution targeting vATPase-B (Milli-Q was used as a negative
control) into a PCR tube and soak the petiole of the green bean primary leaf. 2 hours later, cut three
1 cm × 2 cm leaf pieces from the soaked green bean leaf and place them in three petri dishes with
about 10 thrips. We checked the mortality at 24, 48, 72, and 96 hours[2.1][2.7][2.8]
Test
In particular, there was a large difference in the average mortality rate between
dsRNA-treated and negative control thrips on the third day, which was 8.3% and 31.3%,
respectively. This result confirms the insecticidal effect of indirectly feeding dsRNA
to western flower thrips. We also found that the use of the crude dsRNA samples were as
effective as the reported study with the T7 transcripts.
Learn
Unfortunately, the method examined here did not result in a dramatically high level of
the insecticidal effect. One possible explanation is that the concentration of the dsRNA
solution used in the experiment was too low. We need a more efficient dsRNA production
system.
Cycle4
Construction of a practical insecticidal system for thrips
Construction of a practical insecticidal system for thrips
Design
When considering the practical use of a highly concentrated dsRNA solution as an
insecticide, we can take advantage of the scale merit of mass production. In this case,
our efficient biomolecule production platform "BLOOM" can be used for this purpose (see here for details)
Alternatively, it would be also a promising option to kill thrips if we create transgenic plants that continuously express dsRNA. Our results described above will provide us a smart system to select the most effective dsRNA species for this application.
Alternatively, it would be also a promising option to kill thrips if we create transgenic plants that continuously express dsRNA. Our results described above will provide us a smart system to select the most effective dsRNA species for this application.
References
2.1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020) "Optimization of
dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips
Thrips tabaci", Arch. Insect Biochem. Physiol. 103, e21645.
2.2 Tenllado, F., Martínez-García, B., Vargas, M., and Díaz-Ruíz, J.R. (2003) "Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections", BMC Biotechnol. 3,
2.3 Timmons, L., and Fire, A. (1998) "Specific interference by ingested dsRNA", Nature 395, 854.
2.4 https://2019.igem.org/Team:SZU-China/Experiment
2.5 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018) "Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture", Biotechnol Rep (Amst) 20, e00292.
2.6 Limera, C., Sabbadini, S., Sweet, J.B., and Mezzetti, B. (2017) "New Biotechnological Tools for the Genetic Improvement of Major Woody Fruit Species", Front. Plant Sci. 8, 1418.
2.7 Han, S.H., Kim, J.H., Kim, K., and Lee, S.H. (2019) "Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery", Pestic. Biochem. Physiol. 161, 47–53.
2.8 Singh, S., Gupta, M., Pandher, S., Kaur, G., Goel, N., Rathore, P., and Palli, S.R. (2019) "RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae)", BMC Mol. Biol. 20, 6.
2.2 Tenllado, F., Martínez-García, B., Vargas, M., and Díaz-Ruíz, J.R. (2003) "Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections", BMC Biotechnol. 3,
2.3 Timmons, L., and Fire, A. (1998) "Specific interference by ingested dsRNA", Nature 395, 854.
2.4 https://2019.igem.org/Team:SZU-China/Experiment
2.5 Papić, L., Rivas, J., Toledo, S., and Romero, J. (2018) "Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture", Biotechnol Rep (Amst) 20, e00292.
2.6 Limera, C., Sabbadini, S., Sweet, J.B., and Mezzetti, B. (2017) "New Biotechnological Tools for the Genetic Improvement of Major Woody Fruit Species", Front. Plant Sci. 8, 1418.
2.7 Han, S.H., Kim, J.H., Kim, K., and Lee, S.H. (2019) "Selection of lethal genes for ingestion RNA interference against western flower thrips, Frankliniella occidentalis, via leaf disc-mediated dsRNA delivery", Pestic. Biochem. Physiol. 161, 47–53.
2.8 Singh, S., Gupta, M., Pandher, S., Kaur, G., Goel, N., Rathore, P., and Palli, S.R. (2019) "RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae)", BMC Mol. Biol. 20, 6.
Other applications for cut flower maintenance
We have been investigating various problems in the production, distribution and consumption of
flowers.
Our research has focused on the detection of plant viruses in the production stage. At the same time,
we
also focused on the problem of cut flowers in our daily lives.
-
Cycle1. Commercially available peptide is
expensive
Cycle2. Preparation of peptide in vivo
Cycle3. Improve how to preparate of peptide in
vivo
Cycle4. Run an assay to see how long the flowers
last
Cycle5. Pre-research for measure ability of
peptide to kill bacteria
Cycle6. Disk diffusion test to measure the
ability
of the peptide to kill bacteria
Cycle1.
Commercially available peptide is expensive
Commercially available peptide is expensive
Design
We focused on "NOP-1", which is derived from the NLS of ethylene regulatory factor EIN2, which can
inhibit the senescence signal of flowering plants by acting on ethylene receptors[3.1][3.2].
Currently, the silver thiosulfate complex (STS) is commonly used to extend the life of cut flowers.
However, STS contains silver ion, which is a heavy metal ion, and there are concerns about its
negative
impact on the environment, so there is a need to develop a new alternative method.
One of the causes of cutflowers wilter is the growth of bacteria in the water in the vase, which blocks the conduits of the flowers and prevents the flowers from absorbing water[3.3]. This is not only a big problem at the distribution and sales stage, but also at home.
To solve this problem, we focused on the application of antimicrobial peptides, which have a low environmental impact, instead of antibiotics or chemicals. Antimicrobial peptides act on bacterial cell membranes and kill the bacteria by puncturing the membrane. These peptides have been found to be effective against many bacteria[3.4].
For this experiment, we selected the following three antimicrobial peptides.
Defensin-1
It is an antimicrobial peptide derived from honey bees and is known to be widely effective against Gram-positive bacteria[3.5][3.6].
potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin.
Cecropin A
It is a peptide derived from a moth called Cecropia, which is known to be widely effective against both Gram-positive and Gram-negative bacteria[3.7].
LL37
It is a human-derived peptide that is known to be widely effective against both Gram-positive and Gram-negative bacteria[3.8][3.9].
Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, β-defensins and LL37, produced by human epithelial cells
The first step was to purchase a chemically synthesized peptide to test the effect of this antimicrobial peptide on flower longevity.
One of the causes of cutflowers wilter is the growth of bacteria in the water in the vase, which blocks the conduits of the flowers and prevents the flowers from absorbing water[3.3]. This is not only a big problem at the distribution and sales stage, but also at home.
To solve this problem, we focused on the application of antimicrobial peptides, which have a low environmental impact, instead of antibiotics or chemicals. Antimicrobial peptides act on bacterial cell membranes and kill the bacteria by puncturing the membrane. These peptides have been found to be effective against many bacteria[3.4].
For this experiment, we selected the following three antimicrobial peptides.
Defensin-1
It is an antimicrobial peptide derived from honey bees and is known to be widely effective against Gram-positive bacteria[3.5][3.6].
potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin.
Cecropin A
It is a peptide derived from a moth called Cecropia, which is known to be widely effective against both Gram-positive and Gram-negative bacteria[3.7].
LL37
It is a human-derived peptide that is known to be widely effective against both Gram-positive and Gram-negative bacteria[3.8][3.9].
Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, β-defensins and LL37, produced by human epithelial cells
The first step was to purchase a chemically synthesized peptide to test the effect of this antimicrobial peptide on flower longevity.
Learn
However, chemically synthesized peptides were deemed too expensive to be used in experiments.
Cycle2.
Preparation of peptide in vivo
Preparation of peptide in vivo
Design
Therefore, we decided to express NOP-1 and three antimicrobial peptides in E. coli and purify them.
The
paper was used as a reference to produce the peptide[3.10].
The following method was used[3.11]. The peptide is expressed as a fusion protein: peptide-Mxe GyrA intein-linker-ELK16. The fusion protein aggregates and precipitates due to the action of ELK16. When DTT is added to the precipitated fusion protein, self-cleavage occurs at the N-terminus of the intein, and the peptide is cut out into the water layer.
ELK16
It is an ionic self-assembling peptide that promotes the formation of active inclusion bodies and active protein aggregates in E. coli, and because it self-aggregates, the fusion protein can be recovered as a precipitate.
Mxe GyrA intein
A sequence that is autocatalytically cleaved during protein splicing. By fusing it downstream of the peptide, the peptide can be expressed as a fusion protein, and by adding DTT as a reducing agent, the activity of the intein can be evoked for self-cleavage.
The following method was used[3.11]. The peptide is expressed as a fusion protein: peptide-Mxe GyrA intein-linker-ELK16. The fusion protein aggregates and precipitates due to the action of ELK16. When DTT is added to the precipitated fusion protein, self-cleavage occurs at the N-terminus of the intein, and the peptide is cut out into the water layer.
It is an ionic self-assembling peptide that promotes the formation of active inclusion bodies and active protein aggregates in E. coli, and because it self-aggregates, the fusion protein can be recovered as a precipitate.
Mxe GyrA intein
A sequence that is autocatalytically cleaved during protein splicing. By fusing it downstream of the peptide, the peptide can be expressed as a fusion protein, and by adding DTT as a reducing agent, the activity of the intein can be evoked for self-cleavage.
Build
We made the following plan. First, we inserted the gblock encoding the fusion protein into pET11a to
make a plasmid for peptide production. The plasmid was then transformed into E. coli BL21, and
peptide
production was carried out as described in the design.
Test
After making the plasmid, we produced peptides and cut out the peptides from fusion proteins. The
recovered peptides were electrophoresed on Tricine SDS PAGE to determine whether the peptides were
produced.
1~12 1a~4a,1b~4b,1s~4s (a-sonication 10sx2 ,b-sonication 10s x2 + bioruptor 2sx100, c-sonication
10s x2
+
2sx100)
Learn
The band that is considered to be Def1(5713 Da) appears, while it could not be confirmed as a major
band in the rest of the peptides. Also, the molecular weight of the fusion protein of intein and ELK16
is about 25 kDa, and this band is also visible. It seems that the impurities have been reduced by
additional sonication, but still many impurities remained.
Cycle3
Improve how to preparate of peptide in vivo
Improve how to preparate of peptide in vivo
Design
There are three problems that we found during the first round of peptide purification. First, when the
fusion protein is recovered as a precipitate using ELK16, the purified peptide contains many
impurities
even after washing. Second, the precipitates contain highly viscous material such as genomic DNA,
which
makes it difficult to repeat peptide production because of the time required to wash the
precipitates is long. Third, peptides other than Def1 cannot be identified as major bands, and the
amount
recovered is quite low. To solve this problem, we decided to change the ELK16 part to a His-tag, and
purify the fusion protein of the target peptide and intein by His-tag purification, and then recover
the
peptide by activating the intein by addition of DTT.
The effect of n-end-rule was also considered as the cause of the problem of low recovery[3.12]. I thought that this might have been the reason for the low recovery of peptides other than Def1, so we designed to improve the N-terminal of the peptides. Since NOP1 is originally part of a protein, we will create a plasmid to produce a peptide with valine added to the N-terminus of NOP1.
The effect of n-end-rule was also considered as the cause of the problem of low recovery[3.12]. I thought that this might have been the reason for the low recovery of peptides other than Def1, so we designed to improve the N-terminal of the peptides. Since NOP1 is originally part of a protein, we will create a plasmid to produce a peptide with valine added to the N-terminus of NOP1.
Build
Using an additional primer, a DNA fragment of His tag-intein-peptide was amplified by PCR from the
gblock of ELK16-intein-peptide. Then, we inserted the created fragment into pET11a to make plasmid for
peptide production and purification by His tag. We transformed these plasmids into BL21 and poducted
peptide using the method described in Design.
Test
After plasmids construction was completed, we produced fusion protein in BL21 and pulificated by His
tag.The peptides were electrophoresed by Tricine SDS PAGE.
the bands of the peptides could be seen in Def1 with and without DTT. In all lanes except pet11a, a
band
(of 24 kDa) that seems to be His-intein were visible. Therefore, the expression of the fusion protein
and
the purification of the fusion protein using His tag seems to have been successful. There are three
possible reasons why the other peptides were not identified as bands. First, NOP1 is a particularly
short peptide and may have run away during electrophoresis. The second possibility is that the
peptides were not cleaved by the self-cleavage of the intein, and the third possibility is that the
intein was cleaved at some point before purification and moved to another fraction during imidazole
elution. Def1 was also cut out before the addition of DTT, so this may have occurred the same with
other
peptides.
(1 CecA 2 Def1 3 LL37 4 NOP1 5 NOP1v 6 pet11a 7 CecA 8 Def1 9 LL37 10 NOP1 11
NOP1v)
Cycle4.
Run an assay to see how long the flowers last
Run an assay to see how long the flowers last
Design
An existing method, STS, competitively inhibits ethylene binding to Cu+ in the ethylene receptor
(ETR1)
of flowering plants with Ag+, which also has a high affinity for ethylene. Ag+ is sterically larger
than
Cu+ and occupies the majority of the binding site, allowing it to bind to ethylene and inhibit
ethylene
binding to Cu[3.13].
In contrast, NOP-1 is a peptide known to prolong the life of flowering plants by a mechanism different from that of STS, and can inhibit ethylene-dependent senescence. The signaling pathway triggered by the binding of ethylene to ETR1 inactivates CTR1 kinase and inhibits the phosphorylation of the ethylene regulatory factor EIN2, thereby activating the expression of ethylene response genes. Therefore, NOP-1, a peptide derived from the nuclear localization signal of EIN2, can regulate senescence signaling by binding to the GAF domain of ETR1 and arresting intra- and intermolecular downstream signaling of the receptor. In fact, it was confirmed that flower senescence of flowering plants treated with NOP-1 was suppressed[3.14][1.15].
In contrast, NOP-1 is a peptide known to prolong the life of flowering plants by a mechanism different from that of STS, and can inhibit ethylene-dependent senescence. The signaling pathway triggered by the binding of ethylene to ETR1 inactivates CTR1 kinase and inhibits the phosphorylation of the ethylene regulatory factor EIN2, thereby activating the expression of ethylene response genes. Therefore, NOP-1, a peptide derived from the nuclear localization signal of EIN2, can regulate senescence signaling by binding to the GAF domain of ETR1 and arresting intra- and intermolecular downstream signaling of the receptor. In fact, it was confirmed that flower senescence of flowering plants treated with NOP-1 was suppressed[3.14][1.15].
Build
We tested the effects of (1) NOP-1 and NOP-1v synthesized in vivo, (2) Brassinolide and Brassinazole
given from Mr.Nakano, (3) NOP-1 synthesized chemically. To avoid the possibility of prior treatment
with
STS agents, we used ethylene-sensitive flowering plant, Dianthus, which was purchased in potted form.
With reference to previous studies, 1 mM AgNO3 was used as a positive control and the same buffer in
which each peptide was dissolved was used as a negative control.
Test
No significant flower life extension effect was observed for both NOP-1 synthesized in vivo and NOP-1
synthesized chemically.
Learn
From the literature[3.16], it is known that NOP-1 peptide acts on ethylene
receptors in the endoplasmic
reticulum membrane to prolong the life of cut flowers. In this study, due to seasonal and cost issues,
we used Dianthus instead of roses and carnations, which were used in previous studies. Therefore, the
results of this experiment indicate that in Dianthus, NOP-1 peptide may not reach the ethylene
receptors
of the endoplasmic reticulum membrane in sufficient amounts.
Cycle5.
Pre-research for measuring the ability of peptide to kill bacteria
Pre-research for measuring the ability of peptide to kill bacteria
Design
One of the causes of cut flowers wilter is the growth of bacteria in the water in the vase, which
blocks the conduits of the flowers and prevents the flowers from absorbing water[3.17]. This is not only a
big
problem at the distribution and sales stage, but also at home.
To address this issue, we searched the literature to learn more about the fungal flora in vases. However, we could not find satisfactory previous studies on the fungal flora in vases. Therefore, we decided to investigate what kind of fungi exist in a vase first.
To address this issue, we searched the literature to learn more about the fungal flora in vases. However, we could not find satisfactory previous studies on the fungal flora in vases. Therefore, we decided to investigate what kind of fungi exist in a vase first.
Build
We grew rose, carnation, and delphinium for one week. We had taken bacteria from the vase surface,
stem,
and vase water by platinum loop and smeared on the standard agar medium (Agar 15g,
Glucose 1g, peptone 6g, and Yeast extarct2.5g per 1L water ). In addition, we had collected water in
each place and spread it or its diluted solution on the standard agar medium uniformly by conlarge
stick. This method is appropriate to observe colonies. To compare these plates, we thought that we
could
understand the changes in bacterial flora that depend on time, place, and spices of flowers in the
vase.
Test
The bacterial flora of rose and delphinium had changed over time dramatically. Besides, these flowers
wilted. We could not observe the changes in bacterial flora of carnation as clearly as rose and
delphinium. However, the vase water of carnation cultivated the most amount of bacteria.
Learn
When we observed the changes in bacterial flora, pH also changed. So, the bacteria growing each day
could
not live without the distinctive environment of the day. The dominant species would change as the day
passed[3.18]. One of the major causes of these changes might be that the
dominant species growing rapidly
would decompose the nutrients that plants excrete as a result of the stress reaction or seep out
of
the phloem[3.19][3.20]. This would eliminate waste products, which might result in the continuous environmental
change
in the vase. From these considerations, we thought that if we could always inhibit the bacteria that
might grow in the early stage, we would stop the environmental change. As a result, this would keep
water clean and cut flowers beautiful. In addition, we had researched the bacterial flora in the
stem,
so that we could identify the bacteria that might prevent the vessel from absorbing water. It will be
more
effective to kill the bacteria if we can understand its traits or weak points. If we could do these
experiments, we would be able to change water less often and clean waste water.
Cycle6.
Disk diffusion test to measure the ability of the peptide to kill bacteria
Disk diffusion test to measure the ability of the peptide to kill bacteria
Design
To measure the activity of peptide purified by His-tag in cycle3, we decided to do the disk diffusion.
Build
From pre-research, We found that we were able to measure the activity of Defensin and CecropinA by
using
Bacillus subtilis and E.coli[3.21][3.22][3.23].
Test
We did the disk diffusion method to check if the antimicrobial peptides we made have the ability to
kill
the bacteria[3.24]. In this experiment, we used Escherichia coli and
Bacillus subtilis. However, no antimicrobial activity was observed by our peptides.
Learn
The possible reason for the lack of antimicrobial activity might be that the concentration
of
antimicrobial peptides was not sufficient or the number of bacteria was so large that their activity
could not work sufficiently.
References
3.1 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the
central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep.
9, 1287.
3.2 Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017) "Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control", Front. Plant Sci. 8, 1528.
3.3 Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017) "Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control", Front. Plant Sci. 8, 1528.
3.4 Zhang, L.-J., and Gallo, R.L. (2016) "Antimicrobial peptides", Curr. Biol. 26, R14–R19.
3.5 Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T., and Kobayashi, K. (1990) "A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin", J. Biol. Chem. 265, 11333–11337.
3.6 Defensin1 http://parts.igem.org/Part:BBa_K1104301
3.7 Steiner, H., Andreu, D., and Merrifield, R.B. (1988) "Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects", Biochim. Biophys. Acta 939, 260–266.
3.8 Ouhara, K., Komatsuzawa, H., Yamada, S., Shiba, H., Fujiwara, T., Ohara, M., Sayama, K., Hashimoto, K., Kurihara, H., and Sugai, M. (2005). "Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells", J. Antimicrob. Chemother. 55, 888–896.
3.9 LL-37 antimicrobial peptide http://parts.igem.org/Part:BBa_K1162006
3.10 Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018) "Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein", BMC Biotechnol. 18, 62.
3.11 Lin, Z., Zhao, Q., Zhou, B., Xing, L., and Xu, W. (2015) "Cleavable Self-Aggregating Tags (cSAT) for Protein Expression and Purification. In Insoluble Proteins: Methods and Protocols, E. García-Fruitós, ed", (New York, NY: Springer New York), pp. 65–78.
3.12 Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991) "The N-end rule in bacteria", Science 254, 1374–1377.
3.13 McDaniel, B.K., and Binder, B.M. (2012) "ethylene receptor 1 (etr1) Is Sufficient and Has the Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana", J. Biol. Chem. 287, 26094–26103.
3.14 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep. 9, 1287.
3.15 Binder, B.M. (2020) "Ethylene signaling in plants", J. Biol. Chem. 295, 7710–7725.
3.16 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep. 9, 1287.
3.17 Florack, D.E.A., Stiekema, W.J., and Bosch, D. (1996) "Toxicity of peptides to bacteria present in the vase water of cut roses", Postharvest Biol. Technol. 8, 285–291.
3.18 Simek, K., Pernthaler, J., Weinbauer, M.G., Hornák, K., Dolan, J.R., Nedoma, J., Masín, M., and Amann, R. (2001) "Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir", Appl. Environ. Microbiol. 67, 2723–2733.
3.19 Ratzke, C., and Gore, J. (2018). Modifying and reacting to the environmental pH can drive bacterial interactions. PLoS Biol. 16, e2004248.
3.20 Albert, L.S., and Brown, D.G. (2015). Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria. Colloids Surf. B Biointerfaces 132, 111–116.
3.21 Shen, L., Liu, D., Li, M., Jin, F., Din, M., Parnell, L.D., and Lai, C.-Q. (2012) "Mechanism of action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive bacteria", PLoS One 7, e47194.
3.22 Silvestro, L., Weiser, J.N., and Axelsen, P.H. (2000) "Antibacterial and antimembrane activities of cecropin A in Escherichia coli. Antimicrob", Agents Chemother. 44, 602–607.
3.23 Oren, Z., Lerman, J.C., Gudmundsson, G.H., Agerberth, B., and Shai, Y. (1999) "Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity", Biochem. J 341 ( Pt 3), 501–513.
3.24 Tenover, F.C. (2009) "Antibiotic Susceptibility Testing. In Encyclopedia of Microbiology (Third Edition), M. Schaechter, ed", (Oxford: Academic Press), pp. 67–77.
3.2 Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017) "Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control", Front. Plant Sci. 8, 1528.
3.3 Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017) "Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control", Front. Plant Sci. 8, 1528.
3.4 Zhang, L.-J., and Gallo, R.L. (2016) "Antimicrobial peptides", Curr. Biol. 26, R14–R19.
3.5 Fujiwara, S., Imai, J., Fujiwara, M., Yaeshima, T., Kawashima, T., and Kobayashi, K. (1990) "A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin", J. Biol. Chem. 265, 11333–11337.
3.6 Defensin1 http://parts.igem.org/Part:BBa_K1104301
3.7 Steiner, H., Andreu, D., and Merrifield, R.B. (1988) "Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects", Biochim. Biophys. Acta 939, 260–266.
3.8 Ouhara, K., Komatsuzawa, H., Yamada, S., Shiba, H., Fujiwara, T., Ohara, M., Sayama, K., Hashimoto, K., Kurihara, H., and Sugai, M. (2005). "Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells", J. Antimicrob. Chemother. 55, 888–896.
3.9 LL-37 antimicrobial peptide http://parts.igem.org/Part:BBa_K1162006
3.10 Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018) "Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein", BMC Biotechnol. 18, 62.
3.11 Lin, Z., Zhao, Q., Zhou, B., Xing, L., and Xu, W. (2015) "Cleavable Self-Aggregating Tags (cSAT) for Protein Expression and Purification. In Insoluble Proteins: Methods and Protocols, E. García-Fruitós, ed", (New York, NY: Springer New York), pp. 65–78.
3.12 Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991) "The N-end rule in bacteria", Science 254, 1374–1377.
3.13 McDaniel, B.K., and Binder, B.M. (2012) "ethylene receptor 1 (etr1) Is Sufficient and Has the Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana", J. Biol. Chem. 287, 26094–26103.
3.14 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep. 9, 1287.
3.15 Binder, B.M. (2020) "Ethylene signaling in plants", J. Biol. Chem. 295, 7710–7725.
3.16 Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019) "The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers", Sci. Rep. 9, 1287.
3.17 Florack, D.E.A., Stiekema, W.J., and Bosch, D. (1996) "Toxicity of peptides to bacteria present in the vase water of cut roses", Postharvest Biol. Technol. 8, 285–291.
3.18 Simek, K., Pernthaler, J., Weinbauer, M.G., Hornák, K., Dolan, J.R., Nedoma, J., Masín, M., and Amann, R. (2001) "Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir", Appl. Environ. Microbiol. 67, 2723–2733.
3.19 Ratzke, C., and Gore, J. (2018). Modifying and reacting to the environmental pH can drive bacterial interactions. PLoS Biol. 16, e2004248.
3.20 Albert, L.S., and Brown, D.G. (2015). Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria. Colloids Surf. B Biointerfaces 132, 111–116.
3.21 Shen, L., Liu, D., Li, M., Jin, F., Din, M., Parnell, L.D., and Lai, C.-Q. (2012) "Mechanism of action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive bacteria", PLoS One 7, e47194.
3.22 Silvestro, L., Weiser, J.N., and Axelsen, P.H. (2000) "Antibacterial and antimembrane activities of cecropin A in Escherichia coli. Antimicrob", Agents Chemother. 44, 602–607.
3.23 Oren, Z., Lerman, J.C., Gudmundsson, G.H., Agerberth, B., and Shai, Y. (1999) "Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity", Biochem. J 341 ( Pt 3), 501–513.
3.24 Tenover, F.C. (2009) "Antibiotic Susceptibility Testing. In Encyclopedia of Microbiology (Third Edition), M. Schaechter, ed", (Oxford: Academic Press), pp. 67–77.
Biomolecular Production Platform
Cycle1. Model of APP using usual approaches
Cycle2. Model of APP by adopting a stochastic
approach
Cycle3. Reproduction of 2 plasmid system using
BLOOM
Cycle4. ssrA tag mutagenesis
Cycle5. To derive production efficiency in a
production system using BLOOM
Cycle6. Reproduction of 3 plasmid system using
BLOOM
Cycle7. Plasmid construction
Cycle1.
Model of APP using usual approaches
Model of APP using usual approaches
Design
The efficient production of biomolecules is an important issue for the entire synthetic biology
community. Therefore, we have developed an intelligent biomolecule production system based on
asymmetric
plasmid partitioning.
Multicellular organisms produce functions that cannot be achieved by unicellular organisms through the differentiation and division of roles among different types of cells. We aimed to give E. coli the ability to differentiate and divide its role by using asymmetric plasmid partitioning, and then use this ability to innovate biomolecular production systems.
Asymmetric plasmid partitioning(APP)
Plasmids are normally distributed equally among daughter cells. However, in asymmetric plasmid partitioning, a particular plasmid is distributed to only one daughter cell.
This results in two cell types at each division, one with the plasmid and one without.
The specific mechanism is as follows[4.1].
The two actors are a protein called ParB and a DNA sequence on the plasmid is called ParS. ParS
attaches
to
ParB, and ParB has the property of self-aggregating. Therefore, the plasmid carrying ParS aggregates
under ParB expression, forming a nucleus-like aggregate that can only be distributed to one daughter
cell.
Multicellular organisms produce functions that cannot be achieved by unicellular organisms through the differentiation and division of roles among different types of cells. We aimed to give E. coli the ability to differentiate and divide its role by using asymmetric plasmid partitioning, and then use this ability to innovate biomolecular production systems.
Asymmetric plasmid partitioning(APP)
Plasmids are normally distributed equally among daughter cells. However, in asymmetric plasmid partitioning, a particular plasmid is distributed to only one daughter cell.
This results in two cell types at each division, one with the plasmid and one without.
The specific mechanism is as follows[4.1].
Build
First, we tried to build the first model to understand APP, and tried to simulate APP.
To get started, we classified E.coli as either “stem cells” or “differentiated cells”, and
kinetically calculated averaged plasmid copy numbers of them to reproduce dynamics of the overall
system.
Test
The first model demonstrated only a simple result. It cannot be said the model succeeded in
reproducing
APP.
Learn
Thus, we found that we cannot accurately reproduce APP by using only kinetics, and thought building a
model by using other approaches.
Cycle2.
Model of APP by adopting a stochastic approach
Model of APP by adopting a stochastic approach
Build
We determined to use a stochastic approach in order to reproduce APP, selected “Gillespie
algorithm”[4.2].
We
decided not to simulate dynamics of the overall system or rather, to simulate dynamics of one cell,
and repeated
sufficient times to get the transition of dynamics of the system.
Test
We succeeded in reproducing the change of plasmid copy numbers shown in the graph below. We reproduced
the
result close to the thesis by adjusting the parameters assumed.
Learn
This result suggested that the Gillespie algorithm is efficient. Therefore, we thought that we can
simulate the transition of the concentration of contents based on this model.
Cycle3.
Reproduction of 2 plasmid system using BLOOM
Reproduction of 2 plasmid system using BLOOM
Design
We also thought that it would be more convenient if multiple genes could be expressed in a stepwise
manner in differentiated cells, as in the developmental process of multicellular organisms. Therefore,
we
decided to combine asymmetric plasmid partitioning with two repressor systems to express multiple
genes
in differentiated cells with a time delay.
The specific system is as follows.
The specific system is as follows.
- All cells have both of the two plasmids and are in a stem cell type state. The two genes, GFP and RFP, are both repressed by repressors. However, due to the difference in promoters, there is a difference in concentration between the two repressors.
- When arabinose is added, pBAD is turned on and parB expression is induced.
- The plasmid carrying parS aggregates, and at the next division, asymmetric plasmid partitioning occurs and the plasmid carrying parS drops out from one of the daughter cells, resulting in a differentiated cell.
- In cells that have lost the parS plasmid, no additional repressor production occurs, and the repressor concentration decreases.
- When the concentration of one of the repressors falls below the threshold for repression, the first gene begins to expresing. In this prototype, we have used GFP as the first gene.
- Furthermore, when the concentration of the other repressor falls below the threshold for expression repression, the second gene begins expresing. In this prototype, the second gene is RFP.
- Thus, stepwise gene expression occurs in differentiated cells, and the daughter cells of the one that did not shed the plasmid return to (2), so that this cycle is sustained semi-permanently.
Build
We built a model of the 2 plasmid system. In addition, we investigated what is the most effective
parameter for customizing the time difference by observing the dynamics if we changed a parameter.
Test
The result is as follows.
Please refer to the Modeling page for variation
by degradation rate and promoter strength.
Learn
From the graphs, we found that BLOOM can generate the time difference in expressions of two kinds of
genes, and that the degradation rate of repressors is the most efficient for the time difference.
In addition, because the difference of the promoter activities of repressors makes the difference, we told our wet team the promoter set that generates the time difference maximumly.
In addition, because the difference of the promoter activities of repressors makes the difference, we told our wet team the promoter set that generates the time difference maximumly.
Cycle4.
ssrA tag mutagenesis
ssrA tag mutagenesis
Design
To adapt our BLOOM system to various needs of biomolecule production, we focused on controlling the
time interval between the expression of two genes. Our mathematical modeling shows that the most
important parameter regulating the temporal difference of our BLOOM system is the half-life of
the repressors.
To control the protein half-life, we used ssrA degradation tag. In Escherichia Coli, ssrA-tagged proteins are recognized and efficiently degraded by proteases such as ClpXP or ClpAP. A previous research has identified 12 mutant ssrA tags that promote the degradation of GFP at different rates[4.3]. Therefore, suspecting that there should be more mutants of various degradation efficiencies, we aimed to identify them by mutagenizing WT tag.
To control the protein half-life, we used ssrA degradation tag. In Escherichia Coli, ssrA-tagged proteins are recognized and efficiently degraded by proteases such as ClpXP or ClpAP. A previous research has identified 12 mutant ssrA tags that promote the degradation of GFP at different rates[4.3]. Therefore, suspecting that there should be more mutants of various degradation efficiencies, we aimed to identify them by mutagenizing WT tag.
Build
The wildtype ssrA tag amino acid sequence is AANDENYALAA. It was reported that the three amino acids
at the C terminus of the tag (LAA in wildtype) play a important role on the degradation rate of tagged
protein. Therefore, we chose these three amino acids as the targets for mutagenesis.
For mutagenesis, we used a primer containing three random bases at either one of the target three
C-terminal amino acids (XAA, LXA, LAX), or nine random bases at all of the target three amino acids
(XXX). We also included a primer containing wildtype sequence (LAA), and two reported mutants (AAV &
ASV) for controls.
We fused the ssrA tag sequence to GFP while introducing mutations in the tag by
random-base primers, and cloned the mutant library of ssrA-tagged GFP into a plasmid vector, so that
mutant tags of different activity can be identified by comparing GFP intensity of E.coli
transformants.
Our mutagenesis strategy is as follows:
We chose a low-copy plasmid pSB4K5 as the backbone to be able to compare the fluorescence intensity by trying to avoid the saturation or variation of GFP intensity of colonies.
Our mutagenesis strategy is as follows:
- to J23119 promoter-RBS-GFP insert derived from BBa_K584001, add ssrA tag sequence lacking the three C-terminal amino acids by PCR (first PCR)
- perform the second PCR on the first PCR product to add the rest of the tag sequence while mutagenizing them with different types of random-base primers listed above.
- assemble the second PCR product with a linearized pSB4K5-BBa_J04450 backbone by InFront Assembly (a type of homology arm-mediated seamless cloning like In-Fusion) and transform it into E.coli.
We chose a low-copy plasmid pSB4K5 as the backbone to be able to compare the fluorescence intensity by trying to avoid the saturation or variation of GFP intensity of colonies.
Test
As a result of transformation, colonies of various fluorescence intensity were obtained on the LB
plates. 72 colonies in total were picked from plates, and cultured overnight in LB media. The images
of E.coli cultures were taken under the blue light.
(plate images: under construction)
Bacterial cultures of various GFP intensity were observed.
A10: LB media only, B10: E.coli culture not expressing GFP, B7: E.coli culture expressing non ssrA-tagged GFP, C7: E.coli culture expressing WT ssrA-tagged GFP
Plasmid was extracted from each bacterial culture by miniprep, and then Sanger-sequenced to identify ssrA tag sequence. Furthermore, to quantify protein degradation efficiency of each mutant, GFP Fluorescence of each bacterial overnight culture was imaged on Typhoon 9410 (GE healthcare).
Left: GFP fluorescence image of each bacterial overnight culture
Right: sequenced three C-terminal amino acids of each culture
The mutant ssrA tag sequence of each clone was sequenced, and biological triplicate of each bacterial overnight culture was imaged for GFP fluorescence, including controls (LB media only, bacterial culture not expressing GFP (no GFP), and bacterial culture expressing non ssrA-tagged GFP (no ssrA tag)).
The fluorescence of each culture was quantified by ImageJ as inverted mean gray
value. Measured value of each culture was then normalized to the level of the culture expressing non
ssrA-tagged GFP (no ssrA tag), and presented as mean ± SD from three biological replicates. WT (LAA)
and two mutants reported in a previous research are shown in red boxes.
A10: LB media only, B10: E.coli culture not expressing GFP, B7: E.coli culture expressing non ssrA-tagged GFP, C7: E.coli culture expressing WT ssrA-tagged GFP
Plasmid was extracted from each bacterial culture by miniprep, and then Sanger-sequenced to identify ssrA tag sequence. Furthermore, to quantify protein degradation efficiency of each mutant, GFP Fluorescence of each bacterial overnight culture was imaged on Typhoon 9410 (GE healthcare).
The mutant ssrA tag sequence of each clone was sequenced, and biological triplicate of each bacterial overnight culture was imaged for GFP fluorescence, including controls (LB media only, bacterial culture not expressing GFP (no GFP), and bacterial culture expressing non ssrA-tagged GFP (no ssrA tag)).
Learn
We mutagenized ssrA tag by PCR with mixed primers, and successfully obtained a mutant
collection of engineered ssrA tag derivatives which show various protein degradation efficiencies.
Cycle5.
To derive production efficiency in a production system using BLOOM
To derive production efficiency in a production system using BLOOM
Design
So far, in order to confirm the operation of the production system, the genes of GFP and RFP were used
as the genes to be expressed. Here, in order to discuss the practicality of this system, we decided to
model one of the possible concrete applications of this system.
First of all, we consulted with Ph.D. Ogawa and Ph.D. Takeuchi to get their feedback on how we could use this system in a real microbial factory. Then, we got the idea of recovering the bacteria by agglomerating them and making them bigger, and then searched for a system to realize this.
We decided to use the self-aggregating protein Antigen43, which is known to be expressed on the cell surface and can aggregate cells to form sediments[4.4][4.5][4.6].
First of all, we consulted with Ph.D. Ogawa and Ph.D. Takeuchi to get their feedback on how we could use this system in a real microbial factory. Then, we got the idea of recovering the bacteria by agglomerating them and making them bigger, and then searched for a system to realize this.
We decided to use the self-aggregating protein Antigen43, which is known to be expressed on the cell surface and can aggregate cells to form sediments[4.4][4.5][4.6].
Build
We thought about the system that by incubating E.coli introduced BLOOM with continuous
culturing we can
automatically and permanently collect E.coli in which the product reached equilibrium, and we
calculated
production efficiency of the system.
Test
We derived the condition that we produce the protein more efficiently by using a model of the 2
plasmid
system. In each E.coli, the transition of the concentration of products is shown in the graph
below.
Learn
As a result, we found that our system is 5 times more efficient than conventional systems in producing
in 24 hours.
Cycle6.
Reproduction of 3 plasmid system using BLOOM
Reproduction of 3 plasmid system using BLOOM
Design
In addition to the ParB/ParS pair, it is known that a self-aggregating protein called SopB and a DNA
sequence that binds to SopB called SopC work in a similar manner to cause asymmetric plasmid
partitioning.
Moreover, according to the paper, the parB /parS system and the sopB /sopC system are orthogonal. In other words, even if the ParB/ParS system and the sopB/sopC system are used at the same time, ParB will not be attached to sopC and sopB will not be attached to ParS.
Therefore, we thought that by incorporating sopB/sopC into the above system, we could deepen the hierarchy of the system and establish a more sophisticated division of roles.
Moreover, according to the paper, the parB /parS system and the sopB /sopC system are orthogonal. In other words, even if the ParB/ParS system and the sopB/sopC system are used at the same time, ParB will not be attached to sopC and sopB will not be attached to ParS.
Therefore, we thought that by incorporating sopB/sopC into the above system, we could deepen the hierarchy of the system and establish a more sophisticated division of roles.
- All cells have both of the three plasmids and are in a stem cell type state. 2 genes, GFP and RFP, are both repressed by the repressor, but there is a difference in concentration between the two repressors due to differences in promoters.
- When arabinose is added, pBAD is turned on and parB expression is induced.
- The plasmid with parS aggregates, and at the next division, plasmid asymmetric plasmid partitioning occurs, and the plasmid with parS is shed from one of the daughter cells, and it becomes differentiated cell I.
- In differentiated cell I, the concentration of the first repressor decreases because the production of new repressors does not occur.
- When the concentration of the first repressor falls below the threshold for expression repression, the first gene begins expressing. In this prototype, it is the gene of GFP. In addition to it, sopB expression is also induced at this time.
- The plasmid with sopC aggregates, and at the next division, plasmid asymmetric plasmid partitioning occurs, and the plasmid with sopC is shed from one daughter cell, and becomes differentiated cell II.
- In differentiated cell II, the concentration of the second repressor decreases because the production of new repressors does not occur.
- When the concentration of the second repressor falls below the threshold for expression repression, the second gene begins expressing. In this prototype, it is the gene of RFP.
- Thus, stepwise gene expression occurs in differentiated cells, and the daughter cells that have not shed the plasmid return to (2) or (4), so that the cycle persists semi-permanently.
Build
In order to discuss more complicated systems including APP, we built a model of a 3 plasmid system,
and
simulated the 3 plasmid system.
Test
We succeed in reproducing the phenomenon that two kinds of plasmids are missing in stages as follows.
Learn
Therefore, we demonstrated that APP can compose more complicated systems, and be widely applicable.
Cycle7.
Plasmid construction
Plasmid construction
Build
Plasmid A acts as a reporter of asymmetric plasmid partitioning. It has iRFP and superfolder GFP. We
used BBa_K515005 for the superfolder GFP.
Plasmid B is the second self-aggregating plasmid in the three plasmids system. It has lp-ecfp-sopB, cI
repressor, Double terminator, and sopC. We used J23100 for a promoter.
Plasmid C is the first self-aggregating plasmid in the three plasmids system. It has ParS, Double
terminator, TetR, ip-ecfp-parB. We used J23100 for a promoter.
Plasmid D is self-aggregating in two plasmids system. It has a Double terminator, TetR, cI repressor,
lp-ecfp-parB. We used BBa_B0015 for a Double terminator. Also, we used J23100 and J23114for promoter.
Test
We constructed plasmids based on the improvement considered in the Learn of cycle1.
Learn
We didn't succeed in constructing plasmids B, C, D. So, we compared the number of colonies and
sequences
derived from self-ligated plasmids.
As a result of the research, it turned out that the problem is due to the characteristics of SLiCE that it uses the homology arm to assemble. The principle of SLiCE is almost the same as that of commercial kits such as InFusion HD. Unexpectedly, the NoTl site sequence common to BioBrick's Prefix and Suffix assembled as a homology arm. We concluded that these sequences are added to the plasmid to provide convenience for restriction enzyme cloning. On the contrary, they can be a great obstacle for in vitro assembly. It is a common problem to many iGEM teams and researchers who construct BioBrick using InFusion and Gibson Assembly, so we want them to know about it. Moreover, changed the sequence to attach primers as shown below, reduced the rate of self-ligated plasmids. When you construct plasmids having the same Prefix and Suffix, we strongly recommend that using these primers instead of primers attaching BioBrick's Prefix and Suffix.
As a result of the research, it turned out that the problem is due to the characteristics of SLiCE that it uses the homology arm to assemble. The principle of SLiCE is almost the same as that of commercial kits such as InFusion HD. Unexpectedly, the NoTl site sequence common to BioBrick's Prefix and Suffix assembled as a homology arm. We concluded that these sequences are added to the plasmid to provide convenience for restriction enzyme cloning. On the contrary, they can be a great obstacle for in vitro assembly. It is a common problem to many iGEM teams and researchers who construct BioBrick using InFusion and Gibson Assembly, so we want them to know about it. Moreover, changed the sequence to attach primers as shown below, reduced the rate of self-ligated plasmids. When you construct plasmids having the same Prefix and Suffix, we strongly recommend that using these primers instead of primers attaching BioBrick's Prefix and Suffix.
References
4.1 Molinari, S., Shis, D.L., Bhakta, S.P., Chappell, J., Igoshin, O.A., and Bennett, M.R. (2019) "A
synthetic system for asymmetric cell division in Escherichia coli", Nat. Chem. Biol. 15,
917–924.
4.2 Kazuhide, S. (2004) (JAPANESE) "確率的 シミュレーションアルゴリズムの時間領域量子化", BME Vol.18, No.2
4.3 Flynn, J.M., Levchenko, I., Seidel, M., Wickner, S.H., Sauer, R.T., and Baker, T.A. (2001) "Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis", Proc. Natl. Acad. Sci. U. S. A. 98, 10584–10589.
4.4 Ag43+FLAG+MCS http://parts.igem.org/Part:BBa_K1352001
4.5 Ageorges, V., Schiavone, M., Jubelin, G., Caccia, N., Ruiz, P., Chafsey, I., Bailly, X., Dague, E., Leroy, S., Paxman, J., et al. (2019) "Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation", Sci. Rep. 9, 11100.
4.6 https://2012.igem.org/Team:HokkaidoU_Japan/Project/Aggregation
4.2 Kazuhide, S. (2004) (JAPANESE) "確率的 シミュレーションアルゴリズムの時間領域量子化", BME Vol.18, No.2
4.3 Flynn, J.M., Levchenko, I., Seidel, M., Wickner, S.H., Sauer, R.T., and Baker, T.A. (2001) "Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis", Proc. Natl. Acad. Sci. U. S. A. 98, 10584–10589.
4.4 Ag43+FLAG+MCS http://parts.igem.org/Part:BBa_K1352001
4.5 Ageorges, V., Schiavone, M., Jubelin, G., Caccia, N., Ruiz, P., Chafsey, I., Bailly, X., Dague, E., Leroy, S., Paxman, J., et al. (2019) "Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation", Sci. Rep. 9, 11100.
4.6 https://2012.igem.org/Team:HokkaidoU_Japan/Project/Aggregation