Difference between revisions of "Team:WHU-China/Results"
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<p align="center"> <h2>Figure 2 Gram stain of B. megaterium(left) and E. coli(right).</h2></p> | <p align="center"> <h2>Figure 2 Gram stain of B. megaterium(left) and E. coli(right).</h2></p> | ||
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<p align="center"> <h2>Figure 3 Transformants of RdhANP-pHIS1525(left) and RdhANP-pSPAsp-h(right) on petric plates. </h2></p> | <p align="center"> <h2>Figure 3 Transformants of RdhANP-pHIS1525(left) and RdhANP-pSPAsp-h(right) on petric plates. </h2></p> | ||
Revision as of 03:29, 1 November 2017
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
