Team:Kyoto/Experiments
Experiments
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
5/30 - 6/5
To conduct our detection assays which would be achieved at less price, we first made homemade enzymes for RT-LAMP, which targets Dahlia mosaic virus (DMV). We transformed pMK715 (for BST DNA polymerase), pMK713, and pMK714 (for Reverse Transcriptase) into competent cells, BL21(DE3), and cultivated them in 1L flasks at 37°C then collected cells and stored them at -80°C until needed.
6/20 - 6/26
We purified the proteins using Ni-NTA beads from the frozen cells. The purification was confirmed by SDS-page.
8/11 - 8/13
The purified protein buffer was replaced with a storage buffer by dialysis to create optimal conditions for the enzymes, followed by the confirmation for their activities.
8/16 - 8/22
To prepare the target sequence for our assay, the part of the DMV genome synthesized by IDT was amplified by PCR. The amplification was confirmed by electrophoresis.
8/30 - 9/5
The activity of BST DNA polymerase we prepared was confirmed by a LAMP assay. Since ladders specific to the LAMP reaction were observed, we concluded that it had activity. After this, we used DNA samples to perform the LAMP assay targeting the DMV sequences. Since amplification plots of the sample using the DNA polymerase we prepared were successfully standing up, just like the plot of positive controls, we confirmed that the LAMP reaction for DMV happened.
9/6 - 9/12
We confirmed the activity of RT(p51 and p66 were coupled) we prepared by a reverse transcription assay using the total RNA of yeasts. When the product after the reverse transcription reaction was electrophoresed after PCR, the band was confirmed, indicating that the RT we prepared was active.
9/26 - 10/2
For detection assay, we prepared crRNA for Cas12a by in vitro transcription. We confirmed the transcription by Qubit.
8/22 - 8/28
Plasmid L4440 (pR005), the vector for dsRNA production, was transformed into DH5a, and the plasmid was collected by miniprep. Using this plasmid, we started the construction of plasmids (pR018, pR019, and pR020) to produce dsRNA for RNAi of Arabidopsis thaliana, Ipomoea nil, and Frankliniella occidentalis. First, we amplified the necessary insert part from gblock with two sets of primers. After that, two fragments of the same insert sequence were inserted in different directions into the restriction enzyme-treated pR005 by INFRONT assembly, and transformed into DH5a. The insert was not amplified due to the duplication of the same sequence, so plasmids from multiple colonies were collected by miniprep and treated with restriction enzymes to confirm the insert length. We found that the target PCR fragment was not inserted from the electrophoresis results, so we gave up the plasmid construction using this method and tried another method. 3 types of gblocks were amplified by PCR, and only VATPase and TrxZ were successfully amplified. We ligated the two successful gblocks into pUC118 which were digested by sma1, which were transformed into DH5α.
8/29 - 9/4
Confirming that the target PCR fragments were inserted in the desired direction by colony PCR, the colonies were cultured and the plasmid was collected by miniprep. Collected plasmids and pR005 were treated with restriction enzymes, electrophoresed, purified by Gel purification. These products were ligated to create plasmids pR015 containing VATPase and pR016 containing TrxZ, and transformed into DH5a.The construction of the target plasmids was confirmed by colony PCR, and the plasmids were collected by miniprep. We retired to amplify EPH1, and inserted it in puc118 using the same protocol. Confirming the construction of target plasmid by colony PCR, the plasmid was collected by miniprep. For additional fragment insertions, six plasmids L4440, pR015, pR016, and pUC118 which have three kinds of fragments were treated by restriction enzymes.
9/5 - 9/11
The second fragments of VATPase and Trxz were inserted in the same method as the first fragment insertion. The first fragment of EPH1 was also inserted.
9/12 - 9/18
The constructed plasmids were collected by miniprep. After treatment with restriction enzymes, the insert lengths were examined by electrophoresis. We successfully constructed all plasmids with one insert (pR015, pR016, 017), while we could not confirm the construction of the plasmids with two inserts. Therefore, we will use the plasmid with one insert to produce RNA.
HT115(DE3) was transformed with pR015, pR017, or pR005 (negative control), and cultured in 1 L of LB medium. RNA was collected by ethanol precipitation and electrophoresed, but the desired band was not detected. Therefore, RNA was purified by additional phenol chloroform treatment and electrophoresed with an acrylamide gel to confirm the production of the RNAs to estimate its concentration. Additional RNA production using 6 L of LB medium to obtain the required amount of RNA for the experiment of Frankliniella occidentalis.
HT115(DE3) was transformed with pR015, pR017, or pR005 (negative control), and cultured in 1 L of LB medium. RNA was collected by ethanol precipitation and electrophoresed, but the desired band was not detected. Therefore, RNA was purified by additional phenol chloroform treatment and electrophoresed with an acrylamide gel to confirm the production of the RNAs to estimate its concentration. Additional RNA production using 6 L of LB medium to obtain the required amount of RNA for the experiment of Frankliniella occidentalis.
9/19 - 9/25
We collected RNA with phenol chloroform treatment from the additional culture. We also tried to create RNA by in vitro transcription. Each of the RNAs created this week was electrophoresed with an acrylamide gel to confirm the production of RNA.
<Mortality rate assay of Frankliniella occidentalis>
Using dsRNA targeting vATPase-B, an essential gene of Frankliniella occidentalis, prepared by Hiroto Koga and F. occidentalis grown by Dr. Tokumaru of Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center, we started an RNAi assay to examine the effect of our RNA on the mortality of F. occidentalis. On this day, under the guidance of Dr. Tokumaru, we soaked the petioles of the first green bean leaves in dsRNA solution or Milli-Q. After a few hours, three small leaf fragments were cut out from each green bean leaf, and put with 10 insects in a total of each six petri dishes. The mortality rate will be evaluated after 24, 48, 72, and 96 hours.
<Whitening rate measurement of Arabidopsis thaliana>
We started the RNAi assay to examine the whitening rate of Arabidopsis thaliana using dsRNA targeting the Arabidopsis gene (Trxz) prepared by Hiroto Koga. 200 µl of dsRNA solutions of three different concentrations (0.1, 1, and 10 ng/L) or negative control Milli-Q were added in tubes, and Arabidopsis roots were immersed in each tube. Milli-Q will be periodically replenished and the whitening rate will be measured after one week.
<Mortality rate assay of Frankliniella occidentalis>
Using dsRNA targeting vATPase-B, an essential gene of Frankliniella occidentalis, prepared by Hiroto Koga and F. occidentalis grown by Dr. Tokumaru of Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center, we started an RNAi assay to examine the effect of our RNA on the mortality of F. occidentalis. On this day, under the guidance of Dr. Tokumaru, we soaked the petioles of the first green bean leaves in dsRNA solution or Milli-Q. After a few hours, three small leaf fragments were cut out from each green bean leaf, and put with 10 insects in a total of each six petri dishes. The mortality rate will be evaluated after 24, 48, 72, and 96 hours.
<Whitening rate measurement of Arabidopsis thaliana>
We started the RNAi assay to examine the whitening rate of Arabidopsis thaliana using dsRNA targeting the Arabidopsis gene (Trxz) prepared by Hiroto Koga. 200 µl of dsRNA solutions of three different concentrations (0.1, 1, and 10 ng/L) or negative control Milli-Q were added in tubes, and Arabidopsis roots were immersed in each tube. Milli-Q will be periodically replenished and the whitening rate will be measured after one week.
9/26 - 10/2
<Mortality rate assay of Frankliniella occidentalis>
In the insect RNAi assay started last week, the mortality was evaluated every 24 hours until 96 hours later. In particular, the difference in the mortality rate between dsRNA-treated and Milli-Q treated insects 72 hours after the start of the assay was remarkable, with an average mortality rate of 31.3% for dsRNA-treated and 8.3% for Mili-Q treated insects.
<Whitening rate measurement of Arabidopsis thaliana>
One week later, we evaluated the whitening rate of A. thaliana treated with dsRNA solution or Milli-Q last week, but none of them was bleached. Next, we treated seeds of A. thaliana with a higher concentration (100 ng/µl) of dsRNA and measured the bleaching rate after sprouting. Specifically, we placed a 3 cm square filter paper in the center of each Petri dish, scattered 17 Arabidopsis seeds on the filter paper of the Petri dish, and added 50 µl of 100 ng/µl dsRNA solution or Milli-Q to the paper. We are going to evaluate the bleaching rate when the buds emerge and the leaf length exceeds 5 mm.
In the insect RNAi assay started last week, the mortality was evaluated every 24 hours until 96 hours later. In particular, the difference in the mortality rate between dsRNA-treated and Milli-Q treated insects 72 hours after the start of the assay was remarkable, with an average mortality rate of 31.3% for dsRNA-treated and 8.3% for Mili-Q treated insects.
<Whitening rate measurement of Arabidopsis thaliana>
One week later, we evaluated the whitening rate of A. thaliana treated with dsRNA solution or Milli-Q last week, but none of them was bleached. Next, we treated seeds of A. thaliana with a higher concentration (100 ng/µl) of dsRNA and measured the bleaching rate after sprouting. Specifically, we placed a 3 cm square filter paper in the center of each Petri dish, scattered 17 Arabidopsis seeds on the filter paper of the Petri dish, and added 50 µl of 100 ng/µl dsRNA solution or Milli-Q to the paper. We are going to evaluate the bleaching rate when the buds emerge and the leaf length exceeds 5 mm.
8/8 - 8/14
We started to construct plasmids for peptide production: PET11a-CecA-ELK16, PET11a-Def1-ELK16, PET11a-LL37-ELK16, PET11a-NOP1-ELK16 (pR001-pR004). The PCR products were electrophoresed, and column purified. PET11a was cut with restriction enzymes and assembled with the PCR product or the gblock by INFRONT assembly. Assembled plasmids were transformed into DH5a. The transformed E. coli was plated on amp plates and left overnight to form colonies. Considering the possibility of mutation by PCR, we used plasmid assembled with pet11a and gblock. The construction of the target plasmid was confirmed by colony PCR. The selected colonies were cultured in 3 ml of LB for plasmid collection. The plasmid was collected by miniprep and the sequence was confirmed by Sanger sequencing (we would get the result on 8/14). This plasmid was transformed into E. coli BL21(DE3), which was plated on LB amp to produce the peptide.The results of the sequencing were returned on 8/14, and it was confirmed that all four plasmids we prepared had the correct sequences. Based on these results, the construction of four plasmids from pR001-pR004 were completed.
8/15 - 8/21
BL21(DE3) containing each pR001~pR004 were cultured in 1 L of LB medium, and treated with IPTG to induce peptide expression. The cultured E. coli was collected by centrifugation. These were suspended in Buffer and disrupted by French press and sonication. The peptides were precipitated by centrifugation. After washing the precipitates by Buffer repeatedly, they were divided equally, and DTT was added to one of them to cut out peptides by cleavage of inteins for one day. The difficulty of resuspending the peptides and the results of absorbance measurement of the supernatant after washing (especially CecA) suggested the contamination of genomic DNA. Therefore, the other half of the precipitates was sonicated again with two methods to remove the genomic DNA contamination. After this sonication, DTT was added so that only the peptides became soluble. The peptides were collected separately from the precipitates by centrifugation. The production of the peptides was confirmed by Tricine SDS-PAGE. The gel after electrophoresis was fixed.
<Anti-Bacterial system assay>
We grew rose, delphinium, and carnation for 10 days without exchanging water. We smeared the vase waters on standard agar medium (peptone 5 g, Glucose 1 g, Yeast extract 2.5 g, and Agar 15 g per 1 L water) by platinum loop and cultured their bacteria for one day. We took each water sample from their stem, the edge of the vase, and water.
<Anti-Bacterial system assay>
We grew rose, delphinium, and carnation for 10 days without exchanging water. We smeared the vase waters on standard agar medium (peptone 5 g, Glucose 1 g, Yeast extract 2.5 g, and Agar 15 g per 1 L water) by platinum loop and cultured their bacteria for one day. We took each water sample from their stem, the edge of the vase, and water.
8/22 - 8/28
The gel was stained with CBB decolorized. The band of Def1 was confirmed at the target molecular weight, but for the other peptides, major bands at the peptide molecular weight were not confirmed. In addition, there were a lot of impurities in the samples regardless of whether they had been additionally disrupted or not. Therefore, the peptides were purified by ultrafiltration. We did HPLC to confirm that the target peptides were actually contained. From the results of the HPLC, it was found that the peptides contained a lot of impurities when collected as precipitates using ELK16. Therefore, we decided to collect the peptides using His tag instead of ELK16. In order to prepare the plasmids (pR009-pR012) for this purpose, PCR was performed using new primer sets. The plasmids were constructed using the PCR fragments by the same procedure as before, and the plasmids were collected by miniprep. Considering the possibility that the degradation rate changes depending on the amino acid at the N-terminus, we attached valine to the N-terminus of NOP1 and created primers for creating a plasmid for purification with ELK16 (pR013) or His tag (pR014).
<Anti-Bacterial system assay>
We again grew rose, delphinium, and carnation for one week. To observe the bacteria, the water in each vase did not exchange. The bacteria were collected on the third day and the fifth day by the same method as before. This experiment showed that the bacteria in the vase changed over time. The bacteria found on the third day (day3 bacteria) seemed to make the environment optimal for the growth of bacteria specific to the fifth day (day5 bacteria). After that, day5 bacteria would change the environment optimal for the next bacteria as well. Therefore, we concluded that to keep the vase water clean, it would be more efficient to reduce bacteria on day3 than day7. We decided to use antimicrobial peptides to kill the day3 bacteria. We used HPLC to confirm CecropinA, Defencin1, LL37, and NOP1 purified by ELK16 and extrafiltered. However, we could not detect their presence.
<Anti-Bacterial system assay>
We again grew rose, delphinium, and carnation for one week. To observe the bacteria, the water in each vase did not exchange. The bacteria were collected on the third day and the fifth day by the same method as before. This experiment showed that the bacteria in the vase changed over time. The bacteria found on the third day (day3 bacteria) seemed to make the environment optimal for the growth of bacteria specific to the fifth day (day5 bacteria). After that, day5 bacteria would change the environment optimal for the next bacteria as well. Therefore, we concluded that to keep the vase water clean, it would be more efficient to reduce bacteria on day3 than day7. We decided to use antimicrobial peptides to kill the day3 bacteria. We used HPLC to confirm CecropinA, Defencin1, LL37, and NOP1 purified by ELK16 and extrafiltered. However, we could not detect their presence.
8/29 - 9/4
The same procedure was performed for pR013 and pR014. After collecting pR013 and pR014, we ordered the sequencing of pR009 to pR012 and pR014. From the result, we found that pR010, 011, 012, and 014 were correctly constructed though the length of the linker between the His tag and the intein varied from plasmid to plasmid due to the codon optimization. We ordered additional sequencing of pR009 and pR013 and confirmed that these plasmids were correctly constructed. The 5 plasmids () for his tag purification or PET11a (negative control) was transformed into BL21(DE3) to form colonies. The single colonies were cultured in 1 L of LB medium and induced the expression of fusion proteins by IPTG, and then the cells were collected by centrifugation. The fusion proteins were extracted and purified using His tag.
<Anti-Bacterial system assay>
We cultivated the isolated bacteria from plates on the third and fifth day in liquid medium (peptone 5 g, glucose 1 g, Yeast extract 2.5 g per 1 L water) for one day. After cultivation, we added 300μL of glycerol to 700μL of this liquid culture and kept it at -70℃ as the stock of the bacteria.
<Anti-Bacterial system assay>
We cultivated the isolated bacteria from plates on the third and fifth day in liquid medium (peptone 5 g, glucose 1 g, Yeast extract 2.5 g per 1 L water) for one day. After cultivation, we added 300μL of glycerol to 700μL of this liquid culture and kept it at -70℃ as the stock of the bacteria.
9/5 - 9/11
<Anti-Bacterial system assay>
We again grew rose, delphinium, and carnation for one week in the same condition as before. To understand the bacterial flora deeply, we took third, fifth- and seventh-day’s water samples for NGS analysis. Taking bacteria from the stock, we cultivated them in a liquid medium for 1 day. After that, we did the blocking circle experiment. We made the standard agar medium that includes one kind of bacteria evenly. The paper discs soaked with antimicrobial peptides were put on that plate and cultured for one day. Finally, we measured the diameter of the inhibition zone.
We again grew rose, delphinium, and carnation for one week in the same condition as before. To understand the bacterial flora deeply, we took third, fifth- and seventh-day’s water samples for NGS analysis. Taking bacteria from the stock, we cultivated them in a liquid medium for 1 day. After that, we did the blocking circle experiment. We made the standard agar medium that includes one kind of bacteria evenly. The paper discs soaked with antimicrobial peptides were put on that plate and cultured for one day. Finally, we measured the diameter of the inhibition zone.
9/12 - 9/18
The purified proteins were cutted out by adding DTT to get the target peptides. Electrophoresis on Tricine SDS-PAGE was performed to confirm the production of the peptides. The gel after electrophoresis was fixed.
9/19 - 9/25
CBB staining and decolorization of the gel was performed by the same method as before. The band of Def1 was confirmed at the target molecular weight both before and after the addition of DTT. However, for the other peptides, only bands of intein and ELK16 could be seen, and the other peptide bands could not be confirmed clearly.
<NGS sequencing>
For bacterial flora analysis, phenol chloroform extraction of metagenomic DNA was performed from the flower vase water samples. Following the DNA extraction, qPCR was performed for total DNA quantification, but the result turned out to be a failure. Next, to prepare the NGS library, the first PCR was conducted on the extracted DNA samples with primer sets containing sample-specific barcode sequences. After the first PCR, the PCR products were column-purified and pooled, followed by the second PCR to fuse sequencing adapter sequences. The PCR products from the second PCR were then size-selected by SPRI paramagnetic beads, and sent to a company for next generation sequencing.
<Anti-Bacterial system assay>
We grew rose, delphinium, and carnation for one week in the same condition as before.This time we measured pH of vase water by a pH meter and made plates as well every day. We tried two different ways to culture bacteria. One way is to smear vase water samples by a platinum loop that is the same method as before and the other way is to scatter it on the standard agar medium by a conlarge stick uniformly. In the latter method, we used two different concentration vase water samples. We cultivated Escherichia coli and Bacillus subtilis in a liquid medium for one day and would proceed to the disk diffusion test. Escherichia coli, however, did not multiply, so we used only Bacillus subtilis.
<NOP-1 assay>
Pre-experiments were performed in order to consider the conditions of the assay for evaluating the longevity of flowers. In the experiment, flowers of Arabidopsis thaliana grown by Mr. Yuto Ueda and Mr. Hiroto Koga were used and kept under three conditions (grown with tap water, milliQ, or commercial life extension agent). After that, main-experiments were performed in order to confirm the function of NOP-1(prepared by Mr. Hiroto Koga) and BL・Brz( from Dr. Takeshi Nakano). The flowers used in this experiment were bought at a flower shop in potted form.
<NGS sequencing>
For bacterial flora analysis, phenol chloroform extraction of metagenomic DNA was performed from the flower vase water samples. Following the DNA extraction, qPCR was performed for total DNA quantification, but the result turned out to be a failure. Next, to prepare the NGS library, the first PCR was conducted on the extracted DNA samples with primer sets containing sample-specific barcode sequences. After the first PCR, the PCR products were column-purified and pooled, followed by the second PCR to fuse sequencing adapter sequences. The PCR products from the second PCR were then size-selected by SPRI paramagnetic beads, and sent to a company for next generation sequencing.
<Anti-Bacterial system assay>
We grew rose, delphinium, and carnation for one week in the same condition as before.This time we measured pH of vase water by a pH meter and made plates as well every day. We tried two different ways to culture bacteria. One way is to smear vase water samples by a platinum loop that is the same method as before and the other way is to scatter it on the standard agar medium by a conlarge stick uniformly. In the latter method, we used two different concentration vase water samples. We cultivated Escherichia coli and Bacillus subtilis in a liquid medium for one day and would proceed to the disk diffusion test. Escherichia coli, however, did not multiply, so we used only Bacillus subtilis.
<NOP-1 assay>
Pre-experiments were performed in order to consider the conditions of the assay for evaluating the longevity of flowers. In the experiment, flowers of Arabidopsis thaliana grown by Mr. Yuto Ueda and Mr. Hiroto Koga were used and kept under three conditions (grown with tap water, milliQ, or commercial life extension agent). After that, main-experiments were performed in order to confirm the function of NOP-1(prepared by Mr. Hiroto Koga) and BL・Brz( from Dr. Takeshi Nakano). The flowers used in this experiment were bought at a flower shop in potted form.
9/26 - 10/2
<NOP-1 assay>
We took photos and observed flowers everyday. Interestingly, flowers grown with NOP-1 solution lived 3 days longer than those grown with only the buffer. Although the NOP-1 peptide was thought to be NONE because we could not see it in Tricine SDS-PAGE, we may have actually got it. In that case, the reason why we could not see it in Trisin SDS-PAGE was possibly because the NOP-1 peptide was too short. In order to confirm this hypothesis, we asked Mr. Kento Tamukai to make NOP-1 chemically, and we will do the experiments to compare it with the NOP-1 we prepared.
We took photos and observed flowers everyday. Interestingly, flowers grown with NOP-1 solution lived 3 days longer than those grown with only the buffer. Although the NOP-1 peptide was thought to be NONE because we could not see it in Tricine SDS-PAGE, we may have actually got it. In that case, the reason why we could not see it in Trisin SDS-PAGE was possibly because the NOP-1 peptide was too short. In order to confirm this hypothesis, we asked Mr. Kento Tamukai to make NOP-1 chemically, and we will do the experiments to compare it with the NOP-1 we prepared.
10/3 - 10/9
After ultrafiltration and column purification, mass spectrometry for the correct peptide was performed.
<Anti-Bacterial system assay>
We used HPLC to confirm the existence of peptides purified by His tag.
<NOP-1 assay>
We bought the flower in potted form at the flower shop again. And we did the same experiment again. This time, we increased the number of samples. In addition to this experiment, we started the experiment with NOP-1 which was made chemically.
<Anti-Bacterial system assay>
We used HPLC to confirm the existence of peptides purified by His tag.
<NOP-1 assay>
We bought the flower in potted form at the flower shop again. And we did the same experiment again. This time, we increased the number of samples. In addition to this experiment, we started the experiment with NOP-1 which was made chemically.
8/13 - 8/14
First, we amplified gblocks and biobricks as inserts by PCR. We amplified short inserts by No-template PCR. And we amplified backbone fragments by PCR. We got correct insert fragments but couldn’t get backbone fragments. So, we needed to retry PCR. Also, we needed BBa_I746916 of iGEM 2021 parts as the PCR template for amplifying the fragment of sfGFP, so it was transformed into DH5a and collected the plasmid by miniprep.
8/15 - 8/21
We retried to amplify the fragments that we had failed to do last week. This time, we used linear plasmids treated by restriction enzymes as a template to amplify the backbone. Prof. Makoto Kitabatake, our PI, gave us a plasmid because SopC is derived from F plasmid. We planned to construct plasmids next week.
8/22 - 8/28
After assembly, we transformed plasmids into DH5a competent cells and incubated them overnight. In plasmid A, red and white colonies were observed, so there was a possibility that DpnI digestion was not sufficient. After colony PCR, we got a band in plasmid B at the targeted size, so we read the sequence.
8/29 - 9/11
This week, we improved the protocol for the construction of plasmids. For example, we considered the conditions for PCR and amplified by overlap extension PCR. But despite these improvements, we could not get the targeted plasmids. Moreover, sequencing revealed a point mutation and a small deletion in the plasmid B sequence.And we had to move to another lab, so we started over the construction of the plasmids. All fragments were amplified by PCR. This time, the backbones were linearized with a restriction enzyme and then amplified by PCR. The PCR products were electrophoresed, and column purified.
9/12 - 9/18
The two fragments were fused by OE-PCR and assembled by Infront assembly. Plasmids A, B, and C were transformed into DH5a and incubated overnight. As a result, a large number of colonies were observed in the negative control plates. From this result, it was considered that the template plasmids remained because column purification was insufficient. So, we decided to purify PCR products by gel purification. Using gel-purified PCR products, we did Infront assembly and transformation again.
9/19 - 9/25
By colony PCR, the bands of plasmid A from two colonies were confirmed at the targeted size, so we sequenced these two samples. As a result, these turned out to be the correct sequences. The reason why other plasmids did not succeed seemed to be the large number of inserts. Therefore, to reduce the number of fragments, we amplified three or more fragments by OE-PCR. In addition, the concentration of parB_1 and parB_2 in plasmid B was too low, so we retried PCR. After gel purification, we did Infront assembly and transformation. The result was not successful.
9/26 - 10/2
Based on the results of last week's experiments, we considered the reason why the number of colonies in the negative control was so many. We considered the possibility that the prefix and suffix parts of the backbone sequence were self-ligated. To confirm this hypothesis, we read the sequence of the plasmid gained from the negative control. The results showed that the prefix and suffix parts were self-ligated as we expected, and we needed to redesign the primers. Using the new primers, fragments were amplified by PCR. Since we had all the necessary fragments, we decided to do Infront assembly and transformation again next week.