How are we going to increase the FadL and FadD expression?
Fig 1. Methodoly to overexpression of FadD and FadL genes. At first we cloned all required biobrick in E. coli DH10B to make a stock and then we started the digestions and ligations. We started did the promoters (BBa_I719005/ BBa_I14018/ BBa_R0040) digest with PstI and XbaI and the RBS (BBa_B0034) digestion with SpeI and EcoRI. After that we did gel purified of both parts and then did these two parts ligation, so, we gel purified again and ligate this with a plasmid (PBS1C3) digested with PstI and EcoRI. We did the same way to ligate the FadD coding (BBa_K1076003) or FadL coding (BBa_K1076005) with terminators (BBa_B0015/ BBa_B0010) . After all these steps, we digest the PSB1C3+Promoter+RBS with PstI an XbaI and digest the PSB1C3+Coding+terminator with SpeI and EcoRI and did that ligation and then ligate this ligation in a PSB3C5 digested with PstI and EcoRI and now our vectors are building, how you can see in the figure 1 and 2.
Fig 2. Sequencing construction of to FadD overexpression with 4 diferents promoter(all these contrusction are insert in PSB3C5 plasmid), to compare the best expression result in Shewanella putrefaciens.
Fig 3. Sequencing construction of to FadL overexpression with 4 diferents promoter(all these contrusction are insert in PSB3C5 plasmid), to compare the best expression result in Shewanella putrefaciens. And after that we transform these plasmid in S. Putrefaciens and observed which contrution is better to increase the beta oxidation pathway.