Engineering
In an article from AMB Express in 2019, two key components of degrading guaiacol are mentioned, which are cytochrome P450 (CYP) and Ferredoxin Reductase (FR). We then discovered that plasmids contain genes that express these two substances are respectively WP_085469912 from R.rhodochrous J3 and WP_085469913 from R.rhodochrous J3. Next, we searched for the sequence of these two genes and synthesize them from genetic company.
Figure 1. Linear map of pSEVA424 plasmid and its relevant features. b Structure of the polycistronic operons constructed in pSEVA424. Strains GI, GII, and GIII contain all three gene elements, strain G0 only carries the first gene (CYP gene), and strain GIV contains the first two genes only. Strains GI–III differ by the origin of the ferredoxin gene (WP_085470952 from R. rhodochrous J3 in GI, WP_085469096 from R. rhodochrous J3 in GII and WP_020416430 from Amycolatopsis ATCC 39116 in GIII)
·Problems that we encountered
This plasmid was mainly constructed by Gongze He. According to his response, the construction of this plasmid is relatively successful. We did not encounter any severe problems, and all the works were completed for only one time.
·Experiments & Results
we constructed two plasmids PSB4K5_pTac_CYP_FR (PSHS4) and PSB4K5_pTac_MAO_ALDR (PSHS5) by molecular cloning. We transferred these two plasmids into E. coli BL21, separately. After IPTG induction, we found the target protein expressed, except MAO, by SDS-PAGE.
Figure 4. The process we do in order to determine the expressing of protein.
Figure 5. The analysis of SDS-PAGE of PSHS4 and PSHS5. Lane 1 and 2 are samples of PSHS4 and 5 by overnight LB culture. Lane 3 and 4 are PSHS4 and 5 after overnight culture, and then diluted to M9 and cultured to an OD approximately equal to 0.6. Lane 5 and 6 are PSHS4 and 5 adding IPTG for 3h under 37 degrees centigrade.
We can see the result obviously lane 5 and 6, which can prove that the systems we constructed are successful. However, we found that there are also some protein expressions before induction, which we thought was caused by the leakage of promoter. Moreover, we did not see the obvious expression of MAO, and we will find out the cause and optimize our system in future experiments. In the future, we would determine the activity of enzymes.