Part:BBa_K4192113

Plac-RBS-cdg

This part is used to test the role of cdg (BBa_K4192010) in biofilm production. We used IPTG to induce its expression, and then used crystal violet to dye 96 well plates. The results are shown in the figure below.

The effect of this part is not significant, which means it is hard to work alone. However, it shows obvious enhancement effect when it interacts with gacA gene (BBa_K4192011).

Characterization

We try to increase the expression number of cdg gene in Pseudomonas fluorescens 2P24 and test its role in increasing biofilm production.

We constructed the part BBa_K4192113 and used crystal violet staining to quantitatively test its role in biofilm production. To get more information, please see https://parts.igem.org/Part:BBa_K4192114.

In E.coli, there is no significant difference between recombinant bacteria and wild type, but, it shows obvious yield difference in Pseudomonas fluorescens 2P24.

Fig.1 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.2 Significance analysis of recombinant bacteria

Through Duncan’s multiple range test, there is little difference between wild type and cdg engineered bacteria, and there is also little difference between cdg and gacA groups. The biofilm production of cdg and 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 AgeI site found at 959
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

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