Difference between revisions of "Part:BBa K4192114"

Revision as of 06:51, 13 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 constructed recombinant plasmids pBBR1MCS2-Plac-gacA, pBBR1MCS2-Plac-cdg, pBBR1MCS2-Plac-cdg-gacA respectively. It was transferred into _P. 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℃ 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.

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The biofilm production of _P. fluorescens_ is significantly increased after the recombinant plasmid was transferred. The biofilm production of pBBR1MCS2-Plac-RBS-_cdg_-RBS-_gacA_ recombinant strain is the highest, but the effect of pBBR1MCS2-Plac-_cdg_ recombinant strain is not obviously higher. The average biofilm production of pBBR1MCS2-Plac-_gacA_ recombinant strain is high, but the SD (standard deviation) is large.

During the test, we found that the growth time of *P. 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 calcium ions in the environment could effectively improve the biofilm production from literature^[2].

Therefore, we designed a series of environmental Ca²⁺ gradients and time gradients, and selected the pBBR1MCS2-Plac-RBS-_cdg_-RBS-_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:

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According to the data, the biofilm production tends to the maximum after 26-30 hours of culture, and in the range of 0 M and 0.01 M, the biofilm production increased with the increase of calcium concentration. So we repeat the experiment on the calcium ion, adjust the calcium ion with higher environmental concentration.

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The analysis of the above data shows that the relationship between Ca²⁺ and biofilm production is not linear. Therefore, we chose to control the environmental concentration of Ca²⁺ to 0.01 M and culture for 28 h.

On pBBR1MCS2, there are two promoters upstream of MCS: Plac and PT3. In order to make sure which one is used, we test the effect of induction , we also conducted a group of data comparison of biofilm yield before and after induction.

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We can figure it out by SD analysis, there are no significant difference in blank control and WT (negative control) group, but there shows significant difference among the experimental groups.

Therefore, we carried out the most suitable environmental factors on the production of biofilm: 0.01 M Ca²⁺, 0.5 M IPTG, culture for 28 hours after induced.

1. 1. 1. **Verify the effect of three gene circuits after improving conditions**

The three recombinant strains were re-tested, and the data are shown in the figure below:

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It shows a significant difference in SD analysis as we expected, that both _cdg_ and _gacA_ can increase the concentration of c-di-GMP in bacteria, co-expression can make the concentration of c-di-GMP exceed the threshold, and regulate the production of a larger amont of biofilms.

We can finally conclude that co-express _gacA_ and _cdg_ at the same time can indeed up regulate the expression of bacterial extracellular polysaccharides in _P. fluorescens_ 2P24. However, the condition and effect of separate expression of two genes still needs to be determined by further experiments.