Difference between revisions of "Part:BBa K4192114"

Latest revision as of 16:49, 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^[1] to dye 96 well plates for biofilm testing.

Characterization

At first, we obtained pUC18 linear plasmid by enzyme digestion method, due to its ability to bind with and used the One Step Cloning II Kit (Vazyme Biotech, China) to construct 3 kinds of genetic circuit: pUC18-Plac-gacA, pUC18-Plac-cdg, pUC18-Plac-cdg-gacA respectively for testing and transferred the expression vector into E.coli DH5-alpha by heat shock transformation. Then culture them on a resistant plate containing ampicillin for 10 hours. We cultured positive colony on the resistant LB liquid medium at 200 rpm and 37℃ for 12 hours. After induction, it was inoculated into a 96 well plate to culture for 25 hours, then stained with crystal violet to determine the OD₅₆₀ intensity. Results are shown in the following figure.

We preliminary concluded that the biofilm production of E.coli is very low whether the expression of global regulators is improved or not. The reason might be we used the RBS that inapplicable in E. coli, or the designed genetic circuit is not applicable to E.coli. In conclusion, in order to obtain more reliable verification results, we need to change the recipient bacteria.

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.

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 engineered strain is the highest, but the effect of pBBR1MCS2-Plac-cdg engineered strain is not obviously higher. The average biofilm production of pBBR1MCS2-Plac-gacA engineered 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) engineered 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:

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.

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.

Besides, 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.

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.

After determining the optimal environmental conditions and induction conditions，the three engineered strains were re-tested, and the data are shown in the figure below:

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.

Sequence and Features

References

[1] Cong Liu. Molecular mechanism of biofilm formation in _Shewanella putrefaciens_.2018. China Agricultural University, PhD dissertation

[2] Wang, Tianyang et al. “Magnesium and calcium ions: roles in bacterial cell attachment and biofilm structure maturation.” Biofouling vol. 35,9 (2019): 959-974. doi:10.1080/08927014.2019.1674811