Difference between revisions of "Part:BBa K4192114"

Revision as of 09:29, 5 October 2022

Plac-RBS-cdg-RBS-gacA

This part is used to test the effect of the interaction of cdg gene (BBa_K4192010) and gacA gene (BBa_4192011).As the most important part of promoting Pseudomonas fluorescens to produce biofilm, it has been transferred into E. coli and Pseudomonas fluorescens 2P24 for testing. We used IPTG to induce its expression, and then used crystal violet to dye 96 well plates.

Characterization

We have constructed the parts: BBa_K4192112, BBa_K4192113, BBa_K4192114 by by ClonExpress II one-step cloning kit (Vazyme Biotech, China), and constructed recombinant plasmids pUC18-Plac-gacA, pUC18-Plac-cdg, pUC18-Plac-cdg-gacA. We want to test the effect in E. coli before transferring them to our final chassis bacterium: Pseudomonas fluorescens 2P24.

Transfer three recombinant plasmids into competent E. coli DH5&alpha by heat shock transformation technology. Incubate on LB plate medium containing ampicillin for 12h, verify the positive colony by colony PCR, and use the resistant LB liquid medium to cultivate for 12h at 200rpm and 37℃. Adjust the OD₆₀₀ to the same level, and then inoculate it into 96 well plate. After 20 hours of culture, carry out crystal violet staining, and determine the strength of OD₅₆₀. The data are as follows:

We used wild type E. coli and LB medium as the control group to exclude the influence of the medium itself and bacterial metabolism. It is disappointing that the test effect in E. coli is not ideal. We think that these two regulatory factors cannot play a role in E. coli.

We constructed recombinant plasmids pBBR1MCS-Plac-gacA, pBBR1MCS2-Plac-cdg, pBBR1MCS2-Plac-cdg-gacA by ClonExpress II one-step cloning kit (Vazyme Biotech, China) respectively. It was transferred into Pseudomonas fluorescens 2P24 by electric shock transformation method and cultured on a double resistant plate containing kanamycin and ampicillin for 14 hours. We cultured positive colony on a double resistant LB liquid medium at 200 rpm and 28 degrees for 12 hours. After induction, it was inoculated into a 96 well plate to culture for 28 hours, then stained with crystal violet to determine the OD₅₆₀ intensity. Results are shown in the following figure:

Through Levene's test of equality of error variances, p<0.05, it is proved that there are significant differences between different strains.

The biofilm production of Pseudomonas fluorescens 2P24 is slightly higher than that of E. coli, and the biofilm production is significantly increased after the recombinant plasmid was transferred. The biofilm production of pBBR1MCS2-Plac-cdg-gacA recombinant strain is the highest, but the effect of pBBR1MCS2-Plac-cdg recombinant strain is not obvious. The average biofilm production of pBBR1MCS2-Plac-gacA recombinant strain is high, too, but the error is large.

During the test, we found that the growth time of Pseudomonas fluorescens 2P24 was slow, and the biofilm production of wild type strains was not stable and fluctuated greatly. At the same time, we knew that Ca²⁺ in the environment could effectively improve the biofilm production from literature.

Therefore, we designed a series of environmental Ca²⁺ gradients and time gradients, and selected the pBBR1MCS2-Plac-cdg-gacA (BBa_K4192114) recombinant strain which shows the best effect in the previous test to determine the optimal Ca²⁺ addition amount and culture time. The data are shown in the following figure:

Sequence and Features