Composite

Part:BBa_K4192114

Designed by: Lehan Dong   Group: iGEM22_CAU_China   (2022-09-29)
Revision as of 06:59, 13 October 2022 by OwenDong (Talk | contribs)


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.

<biofilm-2p24-no-ca2.png>
Fig 6. Result of P. fluorescens, without Ca2+, cultured for 28 h.
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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:

<biofilm-of-ca2-and-time.png>
Fig 7. Result of Ca2+ concentration and culture time in P. fluorescens-BBa_K4192114.
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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

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