Team:SCUT China/Demonstrate
Here we have proposed a novel method where the expression levels of multiple genes could be simultaneously regulated without the need to rebuild a library for each system which so-called Versatile Promoter-Toehold Switches (VerProS) pool. We would like to demonstrate the versatility of this approach by using the pool for fine regulation of four genes to enhance the acid tolerant of E. coli complying with all rules and policies approved by the iGEM Safety Committee.
Modification of chassis
In order to complete our project, we have made the necessary modifications to the MG1655. According to our design, we successfully constructed pTarget-placUV5-T7RNAP-rrnBT (Figure1B). After transforming it and pCas9 into MG1655, placUV5-T7RNAP-rrnBT was integrated into the genome of MG1655 (Figure1C), and the new chassis successfully lost the CRISPR-Cas9 system (Figure1D). Subsequently, we have verified whether the T7 polymerase system can be expressed in MG1655. The results indicate that the T7 expression system is compatible with MG1655(Figure1E).
Figure1. Modification of chassis (A) This is the plasmid map of pTarget-X1-LacUV5-RNAP-rrnBT-X2—poxB by Gibson Assembly. (B) Colony PCR gel electrophoresis image. Bands of correct length are seen meaning the construct was successful. (C) When the genome of MG1655 was integrated into placUV5-T7 RNAP, the MGR was transformed into plasmid pET-pT7-RFP. The result of RFP fluorescence values. The photograph of the amount of RFP fluorescence.
Verification of acid tolerant factors
We have constructed pET30-pT7-ybaS, gadC, gadB, katA (Figure 2A, B). And we have tested the ability of each gene to improve the acid tolerant of the chassis. The results showed that biomass of the strains expression of ybaS and gadB increased 53% and 44.2% than that of the initial strain (Figure 2C, D), respectively.
Figure2. Verification of acid tolerant factors (A) This is the plasmid map ofpET30-pT7-ybaS, gadC, gadB, katA by Gibson Assembly. (B) Colony PCR gel electrophoresis image. Bands of correct length are seen meaning the construct was successful. (C) The graph of growth curve of pET30-pT7-gadB. (D) The graph of growth curve of pET30-pT7-ybaS. The y-axis is OD600. The x-axis is time, unit hour. Two hours after the culture of the bacteria, 1.2 μl of 0.05 M IPTG was added to the medium to induce expression. Error bars indicate the standard error of at least three biological replicates. P<0.05.
Improvement of T7 promoters
We successfully constructed the pET30-pT7*-RFP plasmid library with a motif 5 bp random mutation in a specific regions of T7 promoter (Figure 3A). The intensity of the T7 promoter variants (Figure 3C) were defined by ratio of fluorescence value/OD600 using a microplate reader.
Figure3. Improvement of T7 promoters (A) Sequences of core T7 promoter variants. (B)This picture is a plate under fluorescent imaging. There are various mutant strains on this plate. The brightness depends on the strength of the T7 promoter. (C) The graph of ratio of fluorescence value/OD600 of pET30-pT7*-RFP. The y-axis is ratio of fluorescence value/OD600. The x-axis is time, unit hour. Two hours after the culture of the bacteria, 1.2 μl of 0.05 M IPTG was added to the medium to induce expression. The control is E. coli BL21 (DE3). Error bars indicate the standard error of at least three biological replicates. P<0.05.
Construction of VerProS pool
We have completed the construction of 40 plasmids of T7 promoter and trigger combinations. To improve the splicing efficiency of the golden gate assembly, we introduced a negative selection ccdB, which cause a 98.9±0.2% mortality rate (Figure 4A). The positive rate of the transformed strain of the golden gate assembly reaction product was 95.2% (Figure 4B).
Figure4. Construction of VerProS pool (A) These are three plates, pET30 (DH5α), pET30-ccdB (DH5α), pET30-ccdB (transDB3.1). The lethality can be calculated from these plates. (B) Colony PCR gel electrophoresis image. Bands of correct length are seen meaning the construct was successful. (C) The picture of the plates of the product transformed strain.
Construction of working part
We completed the pACYC184-WP by golden gate assembly (Figure 5B). We also successfully integrated the constructed plasmid into E. coli MG1655 T7RNAP. It is then made into competent cells.
Figure5. Construction of working part (A) This is the plasmid map of pACYC184-WP by Gibson Assembly. (B) Colony PCR gel electrophoresis image. Bands of correct length are seen meaning the construct was successful.
Test of VerProS system
We transformed the VerProS pool to engineering bacteria, in which already have the working unit. By enrichment, we obtained the strins library with acid tolerant expression unit. By measuring the growth curve, the biomass of the bacteria increased 143.6% compared to the initial strain. (Figure 6A)
At the same time, we inoculated the bacteria with different survival conditions in the moderate medium (pH=4.5). We then analysis of their growth. And the sequencing of the promoter was obtained by sequencing for modeling (figure 6B).
Further, we attempt to provide a local blast packet designed by ourselves to provide an efficient method for screening promoters from the DNA sequencing result. Teams that try to use VerProS pool can use this blast packet to efficiently filter the final promoter combinations.
Figure6. Test of VerProS system (A) The graph of growth curve of VerProS system. The y-axis is OD600. The x-axis is time, unit hour. Two hours after the culture of the bacteria, 1.2 μl of 0.05 M IPTG was added to the medium to induce expression. Error bars indicate the standard error of at least three biological replicates. P<0.05. (B) The graph of growth curve of VerProS system. The y-axis is OD600. The x-axis is time, unit hour. Two hours after the culture of the bacteria, 1.2 μl of 0.05 M IPTG was added to the medium to induce expression. Error bars indicate the standard error of at least three biological replicates.
Future plan
1. Improve our library capacity through T7 promoter modifications to achieve greater range.
2. Screening more variants to get more data to further optimize our model.
3. Replace the factor of the working part to verify that the VerProS system is reusable.