Team:CAU China/Contribution
Contribution
Parts
A large proportion of our project is based on standard components documented in Part's Registry. As the project processes, we have discovered new information about these existing parts and we are glad to share these learnings with future iGEM teams.
- BBa_K143011 (Promoter gsiB for B. subtilis):
This part used to lack detailed characterization. We have verified that this promoter can respond to salt stress in Corynebacterium glutamicum as well and have added our measurement results to this part according to our experiments. - BBa_K1170003 (p-atp2 in F0F1 ATPase operon from C. glutamicum ATCC 13032):
The original part lacks information in usage and biology, and there had been no characterization to this part yet when we found it in registry. According to literature, we added information about how this promoter responds to pH stress and specified its features. More importantly, we have greatly minimized its sequence and characterized how it works in Corynebacterium glutamicum according to our experiments. - BBa_K2963021 (pgsBCA-encoding a poly-γ-glutamic acid synthetase):
We have noticed that there is a lack of information on the role this part is playing in bacterial capsule and stress resistance. To facilitate the research of future iGEM teams, we added information about other functions of this part. We also supplemented information about its product, γ-polyglutamic-acid, to help future teams discover potential application of this part and its main product.
Others
Besides documentation to existing parts, we have also developed other materials that may help future iGEM teams,among which Educational materials are the most outstanding. We created a series of education materials towards different kinds of audience, aiming to promote synthetic biology. We have already uploaded those materials in bilingual versions and future iGEM teams are welcomed to use and update them freely. Click here to learn more.
You can also download the materials directly:
Handbook for adolescence(English)
Handbook for adolescence(Chinese)
Specific contribution content
BBa_K143011
Characterization
CAU_China 2021 used this part in their composite kill switch to make sure the engineered bacteria would die when the salinity and alkalinity of the soil decrease to an expected level. Since the promoter PgsiB can be activated under mild stress, we use it to respond to salt stress in Corynebacterium glutamicum in our project.
Hence, we aim to verify that the promoter PgsiB, originally from Bacillus subtilis, can respond to salt stress in Corynebacterium glutamicum and we also want to document its response towards different salt concentration.
We use the gene circuit below (BBa_K3796206) to test the function of PgsiB by the fluorescence of GFP.
Fig.1 Gene circuit for PgsiB verification
We build the gene circuit by ClonExpress II one-step cloning kit (Vazyme Biotech, China). Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence.
After sequencing and amplifying, that vector as well as unmodified pXMJ19 were transferred into Corynebacterium glutamicum separately and cultivated on plates. After cultivating them in the LB liquid culture medium at 100 rpm, 30℃ for 12h, the OD600 of the two bacteria were adjusted to nearly the same (nearly 2.0) and inoculated in the salt gradient LB liquid medium. After a cultivation of 24h, the bacterial solution was collected and washed with PBS. Then we measured its fluorescence intensity by HITACHI F-7000, a fluorescence spectrophotometer, according to the excitation light of 488nm and emission light of 507nm and OD600. Data are shown below:
Fig.2 Fluorescence intensity results in the verification of PgsiB (the excitation light of 488nm and emission light of 507nm)
We use the control group(bacteria with empty vector) to exclude the influence of the fluorescence bacteria originally have. Through two-way ANOVA, we know there is significant difference between the control group and the experiment group, proving our results valid. Data shows that the difference of relative fluorescence intensity(fluorescence intensity/OD600) between the two becomes bigger as the salt concentration increase, indicating that there is higher expression of gfp under the control of PgsiB when the salt concentration is high.
We finally come to the conclusion that PgsiB can respond to salinity changes in C. glutamicum, and high salt concentration can improve the expression of the downstream genes of PgsiB. Three parallel experiments were done later, which supported our conclusion. However, we haven’t observed a typical turning point in our curves, and we want to do more experiments about that in near future.
BBa_K1170003
Usage
As an inducible promoter, Patp2 can be used to satisfy the needs for pH responses in project design. For example, it can be used for specifically expressing the target protein in alkaline environment, in which the target protein is often related to the tolerance of high pH environment, like a proton transporter[2]. Also, it can be used together with other pH inducible promoter for a system that can respond and adapt to different pH, from acidic to alkaline.In our project, CAU_China 2021 used this part in their composite kill switch to make sure the engineered bacteria would die when the alkalinity of the soil decrease to an expected level.
Biology
P-atp2 is a part of the FoF1-ATPase operon in Corynebacterium glutamicum. Preliminary studies suggested that C. glutamicum is a moderately alkali-tolerant organism resulted from pH-regulated F0F1 operons which encodes the F0 and F1 multiprotein complexes of the ATP synthase that is involved in the formation of ATP using the electrochemical force of the membrane proton gradient. The genetic organization of the C. glutamicum F0F1 ATP synthase operon maintains the canonical order of the eight structural genes, atpBEFHAGDC.
P-atp2 is the promoter of atpB and its sequence structure is shown in Fig.2. The transcription start points are indicated by bent arrows in the nucleotide sequence. Ribosome-binding sites are shaded in grey, and possible stop codons for the atpI gene are indicated by dashed lines. The -10 and -35 boxes are underlined and in italic letters, and the ATG translation start triplets for atpB, respectively, are shown in bold.[1]
Under alkaline conditions, σH can bind to the P-atp2 promoter of the FoF1-ATPase operon and begin to express downstream genes. When the bacteria grow at alkaline pH, the external pH of the bacteria triggers the change of internal pH, which then acts as an intracellular signal to increase the content of σH factor, so that the binding efficiency of σH with p-atp2 promoter increases and the expression of downstream genes increase as well. Subsequently F0F1 operon expression is induced, thus allowing a higher rate of ATP synthesis and increased growth at its optimal alkaline pH.
Characterization
CAU_China 2021 shortened this part to include only the -35 box, -10 box, and TSS of Patp2 (BBa_K3796204) and characterized its function.We aim to verify that the promoter Patp2 can respond to pH stress in Corynebacterium glutamicum and we also want to document its response towards different alkalinity.
We use the gene circuit below (BBa_K3796207) to test the function of Patp2 by the fluorescence of GFP.
Fig.3 Gene circuit for Patp2 verification
We build the gene circuit by ClonExpress II one-step cloning kit (Vazyme Biotech, China). Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence. After sequencing and amplifying, that vector as well as unmodified pXMJ19 were transferred into Corynebacterium glutamicum separately and cultivated on plates. After cultivating them in the LB liquid culture medium at 100 rpm, 30℃ for 12h, the OD600 of the two bacteria were adjusted to nearly the same(nearly 2.0) and inoculated in the pH gradient LB liquid medium. After a cultivation of 24h, the bacterial solution was collected and washed with PBS. Then we measured its fluorescence intensity by HITACHI F-7000, a fluorescence spectrophotometer, according to the excitation light of 488nm and emission light of 507nm and OD600. Data are shown below:
Fig.4 Fluorescence intensity results in the verification of Patp2 (the excitation light of 488nm and emission light of 507nm)
Through two-way ANOVA, we can know that there’s significant difference between the control group and experiment group under different pH conditions. The relative fluorescence intensity of the control group is relatively stable at the pH range of 7-9.5, while there is a significant increase of relative fluorescence intensity in experimental group. Data shows that the difference of relative fluorescence intensity between the two groups remains high between pH=8.5 and pH=9.0, and reaches its peak near pH=9.0, indicating that the promoter Patp2 has the most ability to enhance its downstream gene expression in this pH range. Also, we can see a sharp drop of the relative fluorescence intensity in the experiment group when the pH is more than 9.5. We assume that this is because C. glutamicum can’t tolerate such a high alkalinity stress and the OD600 decreases a lot, making the relative fluorescence intensity seems abnormal or irregular.
Three parallel experiments were done later, which supported our opinions. We finally come to the conclusion that Patp2 can respond to pH changes in C. glutamicum, and high alkalinity can improve the expression of the downstream genes of Patp2. According to our data, the peak occurs near pH=9.0, which happens to fall in the range of the alkalinity of saline-alkaline soil.
References For Contribution From CAU_China
[1]Barriuso-Iglesias,M. , et al"Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH. " Microbiology (2013).[2] Barriuso-Iglesias, M. , et al. "Transcriptional control of the F0F1 -ATP synthase operon of Corynebacterium glutamicum : SigmaH factor binds to its promoter and regulates its expression at different pH values." Microbial Biotechnology 6.2(2013).
BBa_K2963021
Literature Information
We noticed that there was a lack of information on the Bacterial capsule and stress resistance in the registry. Despite some producting functions about pgsB(capB)、pgsC(capC) and pgsA(capA) , this lack of information will make the future team ignore the application potential of these genes. To facilitate the research of future iGEM teams, we added information about other functions of this part. We also supplemented information about its product.
γ-PGA is the mian product this part synthesize.It was first isolated from the capsule of Bacillus anthracis in 1937. It was found in Bacillus subtilis in 1942 and can be secreted into the culture medium as fermentation product. In both B. subtilis and B. anthracis, the membrane γ-PGA synthetic proteins encoded by the capB,capC,capA operon catalyse synthesis of the capsule polypeptide.
B.subtilis can utilize both D- and L-glutamate as nitrogen sources. D-Glutamate catabolism by this bacterium proceeds after conversion to the L-form by glutamate racemases (the racE and yrpC products). Mutants of racE or yrpC accumulate D-glutamate in latestationary-phase cultures, indicating that B. subtilis cells degrade capsule γ-PGA into its constituent glutamates outside the cells, and utilize them as nitrogen sources during late stationary phase.
Besides, γ-Glutamyltransferase (EC 2.3.2.2; GGT) is widely distributed in nature, from bacteria to animals. For example, independent of the growth phase, Escherichia coli produces GGT in the periplasmic space to utilize γ-glutamylpeptides as amino acid sources.
About γ-PGA and stress resistance:
There are reasons why organisms synthesize a specific secondary metabolite. Why do organisms synthesize γ-PGA?
The main functions of γ-PGA are as follows:
① Protective effect: the cell capsule of B. anthracis contains γ-PGA, which can help bacteria escape phagocytosis, enhance toxicity, make bacteria have non immune characteristics and prevent bacteria from being attacked by cellular antibodies.[9][10] In addition, it can also protect bacteria from phage infection and resist antimicrobial peptides;
② Tolerance to diverse environment: most bacteria synthesize PGA and secrete it into the environment, which can isolate toxic metal ions and reduce the high salt environment, so as to make bacteria tolerate the adverse environment.[11-13]
③ Energy reserve: it can be degraded into glutamic acid for cell growth when there is a lack of energy in the environment.[14]
④ Promote biofilm formation and related to motility.[15]
γ-PGA plays an important role in microbial stress resistance, which has been confirmed by many studies. For example, an research has demonstrated that S. epidermidis secretes poly-γ- DL -glutamic acid (PGA) to facilitate growth and survival in the human host. Importantly, PGA efficiently sheltered S. epidermidis from key components of innate host defense, namely ntimicrobial peptides and neutrophil phagocytosis, and was indispensable for persistence during device-related infection.[10]
These features of γ-PGA provide us with new usage of this part.
References For Contribution From CAU_China
1.Ivanovics G, Bruckner V.1937.Chemical and immunologic studies on themechanism of anthrax infection and immunity. The chemical structure of capsulesubstance of anthrax bacilli and its identity with that of the B. mesentericus[J].Z.Immunitaetsforsch, 90:304-318.
2.Ivanovics G,Bruckner V. 1937.The chemical nature of the immuno-specificcapsule substance of an thrax bacillus[J. Naturwissenschaften,25:250.
3. Bovarnick M. The formation of extracellular D(-)-glutamic acid polypeptideby Bacillus subtillis[J].Journal of Biological Chemistry,145:415-424.
4. Kimura, K. & Itoh, Y. (2003). Characterization of poly-c-glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-c-glutamate. Appl Environ Microbiol 69, 2491–2497.
5. Kimura, K., Tran, L.-S. P. & Itoh, Y. (2004). Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis. Microbiology 150, 2911–2920.
6.Suzuki, H., Kumagai, H. & Tochikura, T. (1986). c-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties. J Bacteriol 168, 1325–1331.
7.Suzuki, H., Hashimoto, W. & Kumagai, H. (1993). Escherichia coli K-12 can utilize an exogenous c-glutamyl peptide as an amino acid source, for which c-glutamyltranspeptidase is essential. J Bacteriol 175, 6038–6040.
8. Kimura K, Tran L S P, Uchida l, et al. 2004. Characterization of Bacillussubtilis v-glut amyltransferase and its involvement in the degradation of capsulepoly--glutamate[J]. Microbiology, 150:4115-4123.
9. Mesnage s,Tosi-Couture E，Gounon P, et al. 1998. The capsule andS-Layer: two independent and yet compatible macromolecular structures in Bacillusan thracis[J].Journal of Bacteriology,180( 1):52-58.
10. Kocianov s, Vuong C, Yao Y, et al. 2005. Key role of poly-γ-DL-glutamicacid in immune evasion and virulence of Staphylococcus epidermidis. The Journalof Clinical Investigation,115:688-694.
11. Mclean R JC，Beauchemin D,Clapham L, et al. 1990.Metal-bindingcharacteristics of the gamma-Glutamyl capsular polymer of Bacillus licheniformis ATCC 9945[J].Applied and Environmental Microbiology,56(12):3671-3677.
12. Kandler o, Konig H, Wiegel J, et al. 1982. Occurrence of Poly-γ-D-Glutamic Acid and Poly-α-L-Glutamine in the Genera Xanthobacter, Flexithrix Sporosarcina and Planococcus[J].Systematic and Applied Microbiology,4:34—-41.
13. Minami H, Suzuki H, Kumagai H, 2004. γ-Glutamyltranspeptidase, but notYwrD,is important in utilization of extracellular glutathione as a sulfur source in Bacillus subtilis[J]. Journal of Bacteriology, 186:1213-1214.
14. Kimura K, Tran L S P, Uchida l, et al. 2004. Characterization of Bacillussubtilis v-glut amyltransferase and its involvement in the degradation of capsulepoly--glutamate[J]. Microbiology, 150:4115-4123.
15. Liu J, He D, Li XZ, et al. 2010.γ-Polyglutamic acid (γ-PGA) produced byBacillus amyloliquefaciens C06 promoting its colonization on fruit surface.International Journal of Food Microbiology,142:190-197.