This year, our team continues the work of Team BBa-K2888010. For the last team, they failed to get protein that can degrade Mycrocystin expressed. We initially think that such failure is due to the deficiency of the gene they choose. As a result, we looked up NCBI and found two species (Novosphingobium sp. THN1 and Sphingomonas sp.) that contain genes that have the similar structure and effects to the one last team employed. As a result, we designed our new parts to handle Mycrocystin: MlrA2 and MlrA3.
Also, we used T7 promoter to replace BBa_J23100 (the promoter last team used) in order to enhance the effects of the expression of our protein. The conservative structure of T7 promoter allowed it to safely combine with most of the protein and thus make it an universal promoter. For the same reason, we replaced BBa-B0015 (the terminator last team used) by T7 terminator.
Furthermore, we employed Condon optimization. By utilizing the preferred Condon inside the E. Coli, we make the process more economic and efficient. The protein will be more easily expressed and the whole experiment would thus be much more stable. The chance of failing will be tremendously reduced.
IntroductionThis BioBrick is an improvement on the one created by the SBS _SH _112144 team (BBa_K2888010). Last year, the team achieved to discover mlrA BBa_K2888010. However, the MlrA was not experimented as membrane protein, causing the results of the protein expression appear to be negative. This year, our team is aimed to successfully express the improved protein, mlrA (), and degrade cyanotoxin released by the lysed cyanobacteria.
Experimental improvementsIn order to express the protein, our team combined the gene of interest with multiple vectors. After running the gel electrophoresis, we found the protein was failed to express in the clear supernatant extract. Last year, SBS _SH _112144 team extracted mlrA gene from Sphingopyxis sp. C-1, an alkalitolerant microcystin-degrading bacterium. This year, the same experiment was also performed on different genes of mlrA selected from different species, ruling out the possibilities of choosing the wrong target genes. The mlrA gene we used in the experiment were extracted from species Novosphingobium sp.THN1 and Shingomonas sp. separately, and were tagged as mlrA2 and mlrA3 for experiment purposes. The result was still negative for protein expression in the supernatant extract.
Table.1 the two rows highlighted in yellow were the mlrA genes used in our team experimented
Through multiple gel electrophoresis, our team discovered a pattern: the protein is always expressed in the sediments. We did some research online and found that, according to Paola Marani’s paper on New Escherichia coli outer membrane proteins, integral membrane proteins cosediment with the outer membrane. Thus, we predicted that mlrA is a membrane protein, which will subside in the sediment along with other lysed cell debris. The sequence of the MlrA gene was analyzed by the website, raptor x prediction. According to Raptor X Predicton, the summary prediction results shows the secondary structure of mlrA2 contains 63% helix, 0% strand and 36% coils.
Figure.1 Summary Prediction Result of MlrA2_SS by Raptor X
Similarly, the secondary structure of mlrA3 contains 64% helix,0% strand and 35% coils.
Figure.2 Summary Prediction Result of MlrA3 by Raptor X
The result confirmed that the secondary structure of this protein is a helix structure within bilayer membrane. According to Francesca M Marassi’s paper, NMR structural studies of proteins, three different polypeptides in oriented bilayers all have been shown to have predominantly helical secondary structure by using multidimensional solution NMR spectroscopy in micelle samples. The analysis of the secondary structure of the target protein appeared to be in accordance with the description in Marassi’s paper. Tertiary structure of the this MlrA gene also accorded with the structure of membrane protein. Therefore, the data supports our prediction that MlrA is a membrane protein.
SBS _SH _112144 team successfully express recombinant plasmids, which contains mlrA and chimeric genes, in DH5-alpha cells. The team transformed and cultured chimeric and mlrA gene in BL21 cells, but they were unable to express MlrA protein in the end. Since our team discovered that mlrA enzyme resides in phospholipid bilayers instead of cytoplasm, we improved the experiment by performing membrane protein purification. We utilized ultracentrifugation to isolate membrane protein from other impurity proteins with 40,000 rpm, whereas the protein located in cytoplasm matrix only need to centrifuge with 12,000 rpm. Our team also added DDM, which is a water soluble nonionic detergent solubilizing membrane proteins to preserve their activity in the membrane-embedded form, into the protein sample. Since the membrane protein precipitate after centrifugation, we used the sediment, instead of the clear supernatant extract, to go through a nickel column. Ultimately, our team used SDS-PAGE Gel to observe that the target protein has expressed, but the expressed amount is few.
According to the results of Gel Electrophoresis, there is a small amount of proteins shown at the target strip. On the other hand, we observed high concentrated protein in the impure proteins that first flowed through the nickel column and in the supernatant after the membrane lysed. The size of the protein was slightly smaller than our target protein. We hypothesized that the protein with higher concentration was probably the MlrA without the sumo tag, otherwise it would just be another impure protein.
Device improvements:The team designs a model that could be easily implemented in water plants to degrade MCLR. The device will be utilized in the last step of tape-water treatment. MlrA will be immobilized on the activated carbon to extract unwanted organic substances from water.
Originally, a layer of activated carbon is set on the bottom of each water tank. Now in order to provide more surface area for reactions between the MlrA and MC-LR to take place, the team improved the device by attaching long rectangular boxes to both sides of the water tank. The boxes contain MlrA, bonded with activated carbon. As water fills the tank, MlrA enzymes in all boxes could work to degrade MC-LR.
Reference:Marani, Paola , et al. "New Escherichia coli outer membrane proteins identified through prediction and experimental verification." Protein Science 15.4(2006):884-889.
Marassi, Francesca M , and S. J. Opella . NMR structural studies of membrane proteins.. NMR of Proteins. Macmillan Education UK, 1993.
Shimizu, Kazuya , et al. "Enzymatic pathway for biodegrading microcystin LR in Sphingopyxis sp. C-1." Journal of bioscience and bioengineering 114.6(2012).