Team:WHU-China/Results
Construction and use of different xylose-inducible expression vectors
We succeeded in construction of several xylose-inducible expression vectors including RdhANP-pSPAsp-hp, RdhANP-pHIS1525, RdhANP-PMP2444, Brick 1-PSB1C3 and Brick 2-PSB1C3, etc. To be honest, we have encountered lots of problems during this step. Take construction of BioBrick 1 as example, we repeated the molecular experiment more than 20 times! Every time when we received empty plate with no transformant on it, we were so disappointed but didn't give up. We hotly discussed with PIs and advisor and checked every single possible problem-occurring steps. Finally, we made it! However, much time was consumed in this step, many other more important steps were delayed as well. But fortunately, with all those efforts, we finally made it and one of the most beautiful result is shown below.
Figure 1 Construction of RdhANP-pSPAsp-hp, RdhANP-pHIS1525.RdhANP was constructed to plasmid PHIS1525 and PSAsp-hp respectively. The restriction enzyme cutting sites of BamH1 and BsrG1 flanked RdhANP through PCR. Then the whole fragment was cut down and combined with carrier by T4 ligase.
Transformation of B. megaterium and protein induction
Besides cloning, we also spent much time in this step. As we’ve introduced in Introduction, though B. megaterium is an ideal host for heterogeneous protein expression, the transformation of it have troubled lots of researchers. At least three different types of transformations have we tried from May to October. Fortunately, we found that this year, FAFU-iGEM was also confronted with this problem because their aimed engineering bacterium is B. megaterium too. It indicates that, once one of us have succeeded in transformation, the other team may also make it by collaboration. And finally, FAFU-iGEM made it, and with their help, we transformed our constructed vectors to B. megaterium and went on the following steps.
Figure 2 Gram stain of B. megaterium(left) and E. coli(right).
Figure 3 Transformants of RdhANP-pHIS1525(left) and RdhANP-pSPAsp-h(right) on petric plates.
Optimization of the RBS
| Parts | Descriptions | Links |
| BBa_K2462003 | Promoters for Bacillus megaterium | http://parts.igem.org/Part:BBa_K2462003 |
| BBa_K2462004 | Promoters for Bacillus megaterium | http://parts.igem.org/Part:BBa_K2462004 |
| BBa_K2462001 | RBS for Bacillus megaterium | http://parts.igem.org/Part:BBa_K2462001 |
| BBa_K2462002 | RBS for Bacillus megaterium | http://parts.igem.org/Part:BBa_K2462002 |
| BBa_K2462006 | Promoter+RBS | http://parts.igem.org/Part:BBa_K2462006 |
Verification of RdhANP-overexpression B. megaterium’s dehalogenation
To verify the expression of RdhANP-pHIS1525 and RdhANP-pSPAsp-hp transformed B. megaterium, we’d first induce the production of RdhANP and then purify it by the His-tag linked to the C-terminal of it. By constructing in vitro proof-to-concept experiment, we’d directly verify RdhANP’s ability to dehalogenate organohalides. However, due to lots of objective limitation, we are not able to confirm its expression and activity by SDS-PAGE and in vitro dehalogenation experiment respectively. But we squeezed our time and tried to demonstrate its activity of dehalogenation by HPLC. In general, RdhANP's activity of dehalogenation can be measured by reduction of substrates or increase of products. We planned to take both methods at first in case of unexpected interference. In the end, we chose to measure the reduction of substrates. Because we've assumed that our measurement of the substrate should be less interfered due to their relatively larger amount.
| Transformed with rdhANP | Untransformed with rdhANP | |
| Induced by xylose | 240.43686/166.5164, 230.89482/166.51640 | 83.32761/194.26079 |
| Uninduced by xylose | 152.95309/81.39657 | -/- |
Table. Values of Peak Area of the peak at 6min of different groups when induced for 0min(after slash) and 120min(before slash).2,6-dichlorophenol(2,6-DCP)(800uM) as substrate and induced at 37℃. Standard Curve indicates that the peak of 2,6-DCP should appear around 6min when stimulated by light of 285.16nm.
Figure 4. Comparison of the output of HPLC with expectation. The Orange line stands for no change of the amount of substrate. Dots colored differently indicate result under different conditions: green for transformed and induced group; blue for untransformed but induced group; yellow for transformed but induced group; red for another repeat.
Unfortunately, just as a saying goes, haste makes waste, the result came out to be disappointing. It just showed us the opposite output to what we’ve expected. The teacher in Testing Center of WHU told us, the strain of B. megaterium we are testing may have produced some proteins that greatly interfere our result. We assume it mainly resulted from three reasons. First, we didn't confirm the expression of RdhANP by SDS-PAGE. Second, the samples we tested haven't been purified enough.Third, B. megaterium might secret something upon the stimulation of added substrate. We still show our ‘seemly wrong’ result above and if time permits, we might do more researches to find out the reasons behind these result.
This year, our team decided to carry on a project using B. megaterium as the host. Through our own experience, we felt that there're few parts in the past years related to the expression in B. mageterium although it's a great host for many proteins to be expressed. Thus, when making parts, we considered more about filling this blank and giving the following teams convenience to use B. megaterium. Unfortunately, we are not able to verify this part successfully, but we do welcome other teams to use our parts and hope our parts can bring them help. As this promoter was from pSPAsp-hp——a plasmid in Addgene, few articles using this promoter can be found on their page for other teams’ reference.
More details about pSPAsp-hp
