We specially searched the iGEM Biological Parts library for related components and picked BBa_R0085, the T7 promoter. This is a biological part submitted by Antiquity in 2005 with only basic DNA sequence information and simple text description information. This part had been improved by iGEM18_ZJU-China in 2018, and the group iGEM18_ZJU-China introduced several mutations into it and improved the yield of target genes. Our team carried out some other mutant T7 promoters, adding data from protein expression in E. coli BL21(DE3).
In addition, through literature research, we found four other new mutant T7 promoter named T7-C1~C4 promoter. We upload the DNA sequence information and basic introduction information in the registry of standard biological parts to provide more choices of protein expression in the prokaryotic system for future iGEM teams.
The T7 promoter is a sequence of DNA 18bp long and it is recognized by T7 RNA polymerase 1. It is a strong promoter in the prokaryotic system and is usually used to induce protein expression in E. coli BL21(DE3) by adding IPTG.
In order to measure the intensity of the mutant promoters, we use Enhance Green Fluorescent Protein (EGFP) as a reporter. EGFP is used as a reporter gene to study gene expression, regulation, cell differentiation, and protein localization. We used the pETD-EGFP plasmid as the PCR template and amplified gene GFP through PCR. The pET28a plasmid was digested with NcoI and BamHI enzymes, and the fragment GFP was inserted into the NcoI and BamHI sites of the pET28a vector through a one-step clone method. Then we transformed the recombinant plasmid into E. coli DH5α competent cells and coated on the LB solid plate. We inoculated the correct colony into the LB culture medium and extracted the plasmid.
In order to introduce mutations into the T7 promoter, we set four different mutants, which were named C1, C2, C3, and C4. We amplified the optimal T7 point mutant plasmid by PCR using pET28a-GFP as the PCR template (Figure 1). DpnI was used to digest templates, then transformed the plasmids to E. coli DH5α competent cells and coated on LB medium plates. Next, we inoculated a mono-colony in LB medium overnight, extracted plasmids, and used Sanger sequencing to verify the successful construction of point mutations.
Figure 1. Gel electrophoresis results of target gene fragments. We transformed the plasmids into BL21(DE3), and measure OD600 and the fluorescence intensity of GFP to determine which type of T7 promotor mutant produces the most. With this method, we can easily find out which mutant T7 promoter is the most efficient to express target proteins. We incubated those four different kinds of bacteria at 37°C for 2.5h. Then we measured the intensity of the EGFP fluorescence (Figure 2). Take the average of each type of T7 promotor mutant as a group, collecting the following data.
Figure 2. validation of the four mutant T7 promoters. 
Figure 3. the intensity of the EGFP fluorescence of the strains
We calculate the A600 of different samples by dividing the average lightness by the average density. From figure 3, we can clearly find that the T7-C1 mutant promotor was the most efficient promoter. So that the promoter T7-C1 could be used to improve the yield of MCFAs in future research on producing MCFAs or other projects.
The gene ydiI (Gene ID: 946190), which is also known as menl, works as 1,4-dihydroxy-2-naphthoyl-CoA hydrolase, and it is conserved in the E. coli MG1655 genome. The E. coli thioesterase YdiI was used as the cycle-terminating enzyme, as it was found to have not only the ability to convert trans-enoyl-CoAs to the corresponding α, β-UCAs, but also a very low catalytic efficiency on acetyl-CoA, the primer and extender unit for the r-BOX pathway. In our project, we overexpressed this enzyme by cloning it into the plasmid pCDFDeut1 to produce MCFAs.
pCDFD-bktB-ydiI is a composite part that contains the key enzymes bktB and ydiI. The plasmid backbone pCDFDeut-1 is usually used for protein expression. It is designed for the co-expression of two target ORFs, and this carrier contains two multiple clone sites, and each site has a T7-lac promoter and a ribosome binding site (RBS).
The gene bktB is a β-ketothiolases that play a key role in the r-BOX cycle and is utilized in the in vivo conversion of Coenzyme A (CoA)-linked precursors such as acetyl-CoA and glycolyl-CoA into β-hydroxy acids.
BBa_K4284033 is a composite part that can be used as a plasmid backbone. we change the DNA sequence of the T7 promoter of the pET28a plasmid by site-direct mutation. In our project, we mutate the promoter to improve the yield of MCFAs, and T7-C1 showed a significant increase in the intensity of the GFP fluorescence, and can be used for future researches.
