Part:BBa_K4192114

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.

Fig.1 96 well plate for biofilm test

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.

Fig.2 Genetic circuit BBa_K4192114 for testing

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:

Fig.3 Biofilm production of E. coli

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.

Then 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:

Fig.4 Comparison between E. coli and Pseudomonas fluorescens 2P24

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:

Fig.5 Biofilm of Ca²⁺ and time

According to the data, the biofilm production tends to the maximum after 26-30h of culture, and it is higher when the environmental concentration of Ca²⁺ is 0.01 M.

Repeat the experiment on the calcium ion group, adjust the Ca²⁺ with higher environmental concentration, and then repeat the experiment:

Fig.6 Biofilm of the concentration of Ca²⁺

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.

Then the three recombinant strains were retested, and the data are shown in the figure below:

Fig.7 Biofilm of Pseudomonas fluorescens 2P24

Through, Levene's test of equality of error variances, p<0.05, it is proved that the experimental results of different recombinant bacteria have significant differences.

Fig.8 Significance analysis of recombinant bacteria

Through Duncan’s multiple range test, there is little difference between wild type and cdg recombinant bacteria, and there is also little difference between cdg and gacA groups. The biofilm production of cdg+gacA recombinant bacteria is significantly higher than that of the other two groups. This shows that the co-expression effect of cdg and gacA genes is better than that of the two genes alone. The analysis shows that both cdg and gacA genes 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 large number of biofilms.

We can draw a conclusion that increasing the copy number of cdg and gacA at the same time can significantly increase the production of biofilm. Related parallel experiments further verify our conclusion. However, the effect of separate expression of two genes still needs to be determined by further experiments.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 468
  Illegal BamHI site found at 459
  Illegal XhoI site found at 557
  Illegal XhoI site found at 1001
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 392
  Illegal NgoMIV site found at 1489
  Illegal AgeI site found at 959
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 410
  Illegal BsaI.rc site found at 977