Team:Hong Kong JSS/Results
Results
Achievement
- We characteristised previous part BBa_K1980002 (iGEM 2016 Oxford) to show it can enhance copper absorption in E. coli
- New parts - Metallothionein from Corynebacterium glutamicum (CgMT) (BBa_K3076100) and showed it can enhance copper absorption in E. coli
- We tested several copper exporter knockout strain of E. coli and found cusF knockout could enhance copper absorption in E. coli
Revisit To Our 2018 iGEM Project
In 2018, we tried to enhance the copper absorption ability of E. coli by ectopically express copper binding metallothionein, the plant Elsholtzia haichowensis EhMT1 and human Homo sapiens MT1A. Our expression vector is pSB1C3 and the E.coli strain was TOP10. The diagram below shows our 2018 parts design.
However, E. coli transformed with pSB1C3-EhMT1 (BBa K2578611)and pSB1C3-MT1A (BBa K2578811) did not show any increase in copper absorption ability. In the copper absorption test, the percentage decrease of copper in both EhMT1 and MT1A transformant are similar to the empty vector control.
It could be due to several reasons, and one of them could be experimental error. We evaluated our experiment procedure in 2018 and identified the following potential errors. This year, we improved and repeat our experiments accordingly:
Our 2018 parts design.
However, E. coli transformed with pSB1C3-EhMT1 (BBa K2578611)and pSB1C3-MT1A (BBa K2578811) did not show any increase in copper absorption ability. In the copper absorption test, the percentage decrease of copper in both EhMT1 and MT1A transformant are similar to the empty vector control.
Results of our 2018 project, we failed to enhance copper absorption ability of the E. coli.
It could be due to several reasons, and one of them could be experimental error. We evaluated our experiment procedure in 2018 and identified the following potential errors. This year, we improved and repeat our experiments accordingly:
- Incubation temperature
Potential error Last year, we incubated the bacterial cultures at room temperature with a magnetic stirrer. The growth and health of the bacteria can be easily affected by the changing temperature. Improvement We improved our experiment by using a shaking incubator at room temperature to 37℃. - Bacteria concentration
Potential error Last year, our experiments are done using bacteria culture grown for 18 hours. However, the growth rate of different bacteria strains can be different and this can affect bacterial concentration. Improvement We controlled the bacteria concentration in our experiment by fixing it to absorbance at 440nm = 1.6. We diluted the bacterial culture with SOB medium to ensure all bacterial cultures were all started in the same concentration. - The composite parts we designed are not working.
- The protein was not expressed properly, it could be due to the fact that E.coli as a prokaryote, cannot fold the eukaryotic protein properly, so there is no functional protein.
However, after we improved our protocol and repeated the experiments, the results are the same. E.coli TOP10 cells transformed with pSB1C3-EhMT1 and pSB1C3-MT1A could not absorb more copper than the empty vector control. We deducted the some reasons for the results:
EhMT1 and MT1A expressing E. coli cannot absorb more copper than normal bacteria.
Characterisation of MymT
Refer to one of the reasons mentioned above, the eukaryotic protein could be expressing improperly. It may be due to the prokaryotic based system in E. coli which is unfavorable for folding eukaryotic protein properly and possibly leads to no functional protein. As a result, a new experiment was carried out for the MymT, a cysteine-rich protein. It has been shown by iGEM16_Oxford Part:BBa_K1980002 that E.coli expressing MymT can absorb more copper.
In our experiment, Mymt was cloned into our pBS1C3 plasmid using the same design as above. We expected the transformed bacteria can absorb more copper. Without a doubt, it reduced the copper level in water by 11.4% after 4 hours. The reduction is significantly different from the control, however, it didn’t reach our expectations.
In our experiment, Mymt was cloned into our pBS1C3 plasmid using the same design as above. We expected the transformed bacteria can absorb more copper. Without a doubt, it reduced the copper level in water by 11.4% after 4 hours. The reduction is significantly different from the control, however, it didn’t reach our expectations.
MymT transformants can absorb more copper than normal bacteria, but the reduction is not very significant.
Improving The Composite Parts Design
In order to obtain a better result, we tried to test for CgMT (BBa K3076100), a new part never been used by any other iGEM team. We also changed the bacterial strain from TOP10 to BL21, specialized from cloning to expressing with a strong T7 promoter.
We utilized pET-151/TOPO vector to drive the expression of CgMT coding sequence under T7 promoter inside BL21(DE) E. coli strain. Then, the bacteria transformants will be tested inside copper (II) sulphate added culture medium to measure their copper absorption efficiency along time.
We utilized pET-151/TOPO vector to drive the expression of CgMT coding sequence under T7 promoter inside BL21(DE) E. coli strain. Then, the bacteria transformants will be tested inside copper (II) sulphate added culture medium to measure their copper absorption efficiency along time.
Our improved composite parts design:
using protein expression strain BL21(DE) and expression vector pET-151/TOPO.
using protein expression strain BL21(DE) and expression vector pET-151/TOPO.
New Parts: CgMT / Vanabin2
Corynebacterium glutamicum metallothionein (CgMT) protein was reported by literature that shows a strong affinity towards binding divalent ions, such as zinc(II) and lead (II). [1] Furthermore, Corynebacterium glutamicum belongs to the same order as Mycobacterium tuberculosis. Previous iGEM team Oxford 2016 demonstrated that MT from M. tuberculosis (BBa K1980002) chelates copper ions in cells effectively, thus we expected CgMT also chelates copper (II) efficiently and it will increase the accumulation of copper ions inside E. coli expressing CgMT.
Based on our results, we showed that after IPTG induction, there is a significant reduction in copper level when compared with IPTG- and empty vector transformant control. It indicates E. coli strains (BL21) expressing CgMT has increased copper absorption ability.
In the graph, the percentage change of copper(II) ion concentration along time is shown. The copper added media were incubated with IPTG+ (IPTG induced CgMT expression E. coli), IPTG- (CgMT expression E. coli without IPTG added) and Empty (Empty vector control E. coli). IPTG+ showed ~30% decrease, IPTG- showed ~18% decrease and empty vector control showed ~14% decrease.
On the other hand, when we compare IPTG- with empty vector control, IPTG- also showed a statistical significant difference. We believe it could be due to the leaky expression of the T7 promoter. Further investigation is required.
We aimed to increase the amount of copper accumulation in E. coli so we studied the effect of knocking out some of the major copper exporter genes inside the bacterial genome. The National Institutes of Genetic in Japan has provided us with the 4 E. coli knockout strains, cusA, cusF, CopA and cutA. [3-4]
All these strains contain a single-gene deletions in E.coli K-12. [5] As these genes are responsible for the export of copper ion from the cell, we expect more copper ion will be retained inside of the cell once these genes are knockout.
After the knockout strains were tested in the bacterial copper absorption test, cusA, copA and cutA knockout strains do no show any increased copper absorption ability when compared to the control (normal E. coli). This could be due to the fact that the gene has not been completely nullified or gene redundancy, whereas the existence of multiple genes in the genome performing the same function.
However, for cusF knockout strain, we can see the copper level was reduced by 19% after 2 hours of incubation and the reduction increased to 27% after 4 hours. it is significantly higher than that of the control, which is 9% at 2 hours and 17% at 4 hours. This is implicating that after cusF was knocked out, the bacteria failed to export copper and thus retaining more copper ions inside the cell.
In our experiments, we did not monitor the expression level of CgMT. This is because we do not have the equipment and reagents for western blot. Also, due to the extremely hazardous nature of acrylamide, we are not suggested to conduct western blotting in our high school laboratory. We are currently seeking collaboration with local university so that we can use their facilities to perform protein work.
In our experiments, we had demonstrated that ectopic expression of CgMT and knockout of cusF in E. coli can increase the copper adsorption ability. Compared to the empty vector control, more copper from the solution is removed when mixing with these GM E. coli. However, due to time constraint, we have not compared the effect of having CgMT expressed in the cusF knockout background. We believe this could further increase the copper adsorption ability of E. coli.
Reference:
Based on our results, we showed that after IPTG induction, there is a significant reduction in copper level when compared with IPTG- and empty vector transformant control. It indicates E. coli strains (BL21) expressing CgMT has increased copper absorption ability.
The percentage decrease of copper concentration in the culture medium.
In the graph, the percentage change of copper(II) ion concentration along time is shown. The copper added media were incubated with IPTG+ (IPTG induced CgMT expression E. coli), IPTG- (CgMT expression E. coli without IPTG added) and Empty (Empty vector control E. coli). IPTG+ showed ~30% decrease, IPTG- showed ~18% decrease and empty vector control showed ~14% decrease.
On the other hand, when we compare IPTG- with empty vector control, IPTG- also showed a statistical significant difference. We believe it could be due to the leaky expression of the T7 promoter. Further investigation is required.
Gene Knockout experiments
We aimed to increase the amount of copper accumulation in E. coli so we studied the effect of knocking out some of the major copper exporter genes inside the bacterial genome. The National Institutes of Genetic in Japan has provided us with the 4 E. coli knockout strains, cusA, cusF, CopA and cutA. [3-4]
All these strains contain a single-gene deletions in E.coli K-12. [5] As these genes are responsible for the export of copper ion from the cell, we expect more copper ion will be retained inside of the cell once these genes are knockout.
Genes related to exporting copper ions from the cell.
Knockout strains of cusA, cusF, copA and cutA were tested for the copper absorption ability.
Knockout strains of cusA, cusF, copA and cutA were tested for the copper absorption ability.
After the knockout strains were tested in the bacterial copper absorption test, cusA, copA and cutA knockout strains do no show any increased copper absorption ability when compared to the control (normal E. coli). This could be due to the fact that the gene has not been completely nullified or gene redundancy, whereas the existence of multiple genes in the genome performing the same function.
Knockout of cusA, copA and cutA cannot increase copper absorption ability of E. coli.
However, for cusF knockout strain, we can see the copper level was reduced by 19% after 2 hours of incubation and the reduction increased to 27% after 4 hours. it is significantly higher than that of the control, which is 9% at 2 hours and 17% at 4 hours. This is implicating that after cusF was knocked out, the bacteria failed to export copper and thus retaining more copper ions inside the cell.
Knockout of cusF significantly increase copper absorption ability of E. coli.
Limitation and Improvement
In our experiments, we did not monitor the expression level of CgMT. This is because we do not have the equipment and reagents for western blot. Also, due to the extremely hazardous nature of acrylamide, we are not suggested to conduct western blotting in our high school laboratory. We are currently seeking collaboration with local university so that we can use their facilities to perform protein work.
Future Direction
In our experiments, we had demonstrated that ectopic expression of CgMT and knockout of cusF in E. coli can increase the copper adsorption ability. Compared to the empty vector control, more copper from the solution is removed when mixing with these GM E. coli. However, due to time constraint, we have not compared the effect of having CgMT expressed in the cusF knockout background. We believe this could further increase the copper adsorption ability of E. coli.
Reference:
- Jafarian, V., & Ghaffari, F. (2017). A unique metallothionein-engineered in Escherichia coli for biosorption of lead, zinc, and cadmium; absorption or adsorption? Microbiology, 86(1), 73–81. doi: 10.1134/s0026261717010064
- Rensing, C., Fan, B., Sharma, R., Mitra, B., & Rosen, B. P. (2000). CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. Proceedings of the National Academy of Sciences of the United States of America, 97(2), 652–656. doi:10.1073/pnas.97.2.652
- Padilla-Benavides, T., George Thompson, A. M., McEvoy, M. M., & Argüello, J. M. (2014). Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF. The Journal of biological chemistry, 289(30), 20492–20501. doi:10.1074/jbc.M114.577668
- Franke, S., Grass, G., Rensing, C., & Nies, D. H. (2003). Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. Journal of bacteriology, 185(13), 3804–3812. doi:10.1128/jb.185.13.3804-3812.2003
- Fong, S. , Camakaris, J. and Lee, B. T. (1995), Molecular genetics of a chromosomal locus involved in copper tolerance in Escherichia coli K‐12. Molecular Microbiology, 15: 1127-1137. doi:10.1111/j.1365-2958.1995.tb02286.x
- Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular systems biology, 2, 2006.0008. doi:10.1038/msb4100050