Difference between revisions of "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) i. 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. ii. The photograph of the amount of RFP fluorescence.

Verification of acid tolerant factors

We have built pET30-pT7-ybaS, gadC, gadB, katA (figure2B). And we have tested the ability of each gene to improve the acid tolerant of the chassis. The results showed that ybaS and gadB increased by 59.6% and 44.2% (figure2C, 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 and 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 by synthesizing primers containing five random mutated base sequences of specific regions of T7 promoters (figure3A). The intensity of each mutant promoter (figure 4C) was defined by obtaining a ratio of fluorescence value/OD600 by 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. And in order to improve the splicing efficiency of the golden gate assembly, we constructed a final vector of pet30-ccdB with a 98.9±0.2% mortality rate (figure4A). The positive rate of the transformed strain of the golden gate assembly reaction product was 95.2% (figure4B). These are the plates of the product transformed strain (figure4C).

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 construction of pACYC184-WP with the golden gate assembly (figure5B). And we have successfully integrated it into the chassis system. 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 which have the working part. By enrichment, we obtained the acid tolerant bacteria of the optimal promoter arrangement (figure6A). By measuring the growth curve, the bacteria increased by 144.6% compared to the chassis organism.
At the same time, we inoculated the bacteria with different survival conditions in the medium under the pH=4.5 plate. We then measured their de growth curves. 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.

Future plan

1. Improve our library capacity through T7 promoter modifications to achieve greater range.
2. Get more data to further optimize our model.
3. Replace the factor of the working part to verify that the VerProS system is reusable.