Team:WHU-China/Results

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Results

• 1 Construction and use of different xylose-inducible expression vectors
• 2 Transformation of B. megaterium and protein induction
• 3 Optimization of the RBS
• 4 Verification of RdhANP-overexpression B. megaterium’s dehalogenation

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 2-PMP2444 and Brick 2-pSPAsp-hp. 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 had no idea about the reason for the failures but had to repeat again and again. So, much time was consumed in this step, many other more important steps were delayed. 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. megateium is an ideal host for heterogeneous protein expression, 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 in fronted 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 N-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. 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. Maybe there’s something wrong with our induction of protein production. And 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. Below we still show our ‘seemly wrong’ result and if time permits, we might do more researches to find out the reasons behind these result.

B. megaterium	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	120min/0min

Table. Values of Peak Area of the peak at 6s of different groups when induced for 0min(after slash) and 120min(before slash)

    3,5-dichloro-2-hydroxybenzoic acid as substrate and induced at 17℃. Standard Curve indicates that the peak of 3,5-dichloro-2-hydroxybenzoic acid should appear around 6s when stimulated by light of 285.16nm.

Figure 4. Comparison of output of HPLC with expectation.

     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.