Difference between revisions of "Part:BBa K4192011"
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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. </p>
 
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. </p>
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[[File:CAUChinabiofilm2P24fin.png|600px|thumb|center|]]
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[[File:CAUChinabiofilm2P24fin.png|300px|thumb|center|]]
 
<center><strong> Fig.2 Significance analysis of recombinant bacteria </strong></center>
 
<center><strong> Fig.2 Significance analysis of recombinant bacteria </strong></center>
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<p>Through Duncan’s multiple range test, there is 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. </p>
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<p>According to the results of ANOVA(Duncan's method), there is little difference between <i>cdg</i> and <i>gacA</i> groups. The biofilm production of co-express <i>cdg</i> and <i>gacA</i> bacteria is significantly higher than that of the other two groups. This shows that the co-expression effect of <i>cdg</i> and <i>gacA</i> genes is better than that of the two genes alone. The analysis shows that both <i>cdg</i> and <i>gacA</i> 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 larger number of biofilms. </p>
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<p>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. </p>
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<p>We can draw a conclusion that increasing the copy number of <i>cdg</i> and <i>gacA</i> 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. </p>
  
 
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<!-- Add more about the biology of this part here

Revision as of 15:36, 12 October 2022


gacA from Pseudomonas fluorescens str. 2P24

It encoding global regulator GacA. It can enhance extracellular polysaccharide (EPS) synthesis, enhance antifungal ability and weaken exercise ability[1]. Therefore, when it is co-expressed with other regulatory genes, the production of biofilm can be significantly increased. It belongs to a two-component regulatory system, typically composed of a membrane-bound sensor kinase (GacS) and a cytoplasmic response protein (GacA), distribute widely in bacteria. A number of publications demonstrated that the GacS/GacA system globally regulated the production of the extracellular compounds and exoenzymes and played an important role in the biocontrol activity of the Pseudomonas spp[2]. GacA can regulate a variety of downstream physiological activities, so the effect is not single. It even increase the content of c-di-GMP, similar to the cdg gene (BBa_K4192010).

Characterization

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

We connected it to the downstream of Plac to obtain the part BBa_K4192112 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.

CAUChinabiofilm2P24.png
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.

CAUChinabiofilm2P24fin.png
Fig.2 Significance analysis of recombinant bacteria

According to the results of ANOVA(Duncan's method), there is little difference between cdg and gacA groups. The biofilm production of co-express cdg and gacA 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 larger 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 316
  • 1000
    COMPATIBLE WITH RFC[1000]



References

[1]Seaton, Sarah C et al. “Pleiotropic effects of GacA on Pseudomonas fluorescens Pf0-1 in vitro and in soil.” Applied and environmental microbiology vol. 79,17 (2013): 5405-10. doi:10.1128/AEM.00819-13

[2]Yan Xiaoxue.The role of regulatory gene gacA in the biocontrol activity of Pseudomonas fluorescens 2P24.2004.Northwest Sci-Tech University of Agriculture and Forestry,PhD dissertation..