Difference between revisions of "Part:BBa K4284016"

Revision as of 13:55, 26 September 2022

T7-C3 promoter

T7-C2 promoter

contribution

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.

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 a new mutant T7 promoter named T7-C3 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.

Figure 1. The map of Site-direct mutation of the T7-C3 promoter..

Construction of T7-EGFP plasmids

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.

a) Site-direct mutation of the T7 promoter

In order to introduce mutations into the T7 promoter, we set four different mutants, which were named C3, We amplified the optimal T7 point mutant plasmid by PCR using pET28a-GFP as the PCR template (Figure 2). 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 2. Gel electrophoresis results of target gene fragments. M: DNA Marker. C1: The gene fragment T7-C1 mutant promoter plasmid, correct C2: The gene fragment T7-C2 mutant promoter plasmid, correct C3: The gene fragment T7-C3 mutant promoter plasmid, correct C4: The gene fragment T7-C4 mutant promoter plasmid, correct..

b) Screening for optimal mutations

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 3). Take the average of each type of T7 promotor mutant as a group, collecting the following data.

Figure 3. validation of the four mutant T7 promoters. a. T7-C1 mutant promoter strain b. T7-C2 mutant promoter strain c. T7-C3 mutant promoter strain d. T7-C4 mutant promoter strain..

Figure 4. 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 4, we can clearly find that the T7-C3 mutant promotor was more efficient than T7 WT promoter.

Sequence and Features