Difference between revisions of "Part:BBa K4192010"
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<partinfo>BBa_K4192010 short</partinfo>
 
<partinfo>BBa_K4192010 short</partinfo>
  
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The cdg (BBa_K4192010) encodes the DGC protein of <i>Pseudomonas fluorescens</i>,also named as <i>dgc</i>. DGC protein catalyzes the synthesis of r c-di-GMP, which is an important regulator<sup>[1]</sup>. It can promote bacterial adhesion, extracellular polysaccharide synthesis and other functions.
+
The cdg (BBa_K4192010) encodes the CDG protein (aka DGC) of <i>Pseudomonas fluorescens</i>,also named as <i>dgc</i>. CDG protein catalyzes the synthesis of r c-di-GMP, which is an important regulator<sup>[1]</sup>. It can promote bacterial adhesion, extracellular polysaccharide synthesis and other functions.
 
Increasing its expression can increase the production of biofilm.  
 
Increasing its expression can increase the production of biofilm.  
  
 
===Characterization===
 
===Characterization===
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<p>We try to increase the expression number of <i>cdg</i> gene in Pseudomonas fluorescens 2P24 and test its role in increasing biofilm production.</p>
+
<p>We try to increase the expression number of <i>cdg</i> gene in <i>Pseudomonas fluorescens</i> 2P24 and test its role in increasing biofilm production.</p>
 
<p>We connected it to the downstream of Plac to obtain 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.</p>
 
<p>We connected it to the downstream of Plac to obtain 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.</p>
 
<p>In <i>E.coli</i>, there is no significant difference between recombinant bacteria and wild type, but, it shows obvious yield difference in <i>Pseudomonas fluorescens</i> 2P24.
 
<p>In <i>E.coli</i>, there is no significant difference between recombinant bacteria and wild type, but, it shows obvious yield difference in <i>Pseudomonas fluorescens</i> 2P24.
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<center><strong> Fig.1 Biofilm of <i>Pseudomonas fluorescens</i> 2P24 </strong></center>
 
<center><strong> Fig.1 Biofilm of <i>Pseudomonas fluorescens</i> 2P24 </strong></center>
  
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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>
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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>
 
[[File:CAUChinabiofilm2P24fin.png|600px|thumb|center|]]
 
[[File:CAUChinabiofilm2P24fin.png|600px|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 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.</p>
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<p>Through Duncan’s multiple range test, there is little difference between wild type and <i>cdg</i> engineered bacteria, and there is also little difference between <i>cdg</i> and <i>gacA</i> groups. The biofilm production of co-express <i>cdg</i> and <i>gacA</i> engineered 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 large 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>
+
<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>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 15:48, 12 October 2022


cdg gene from Pseudomonas fluorescens str.2P24

The cdg (BBa_K4192010) encodes the CDG protein (aka DGC) of Pseudomonas fluorescens,also named as dgc. CDG protein catalyzes the synthesis of r c-di-GMP, which is an important regulator[1]. It can promote bacterial adhesion, extracellular polysaccharide synthesis and other functions. Increasing its expression can increase the production of biofilm.

Characterization

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

We connected it to the downstream of Plac to obtain 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.

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

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 co-express cdg and gacA engineered 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 263
    Illegal BamHI site found at 254
    Illegal XhoI site found at 352
    Illegal XhoI site found at 796
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 187
    Illegal AgeI site found at 754
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 205
    Illegal BsaI.rc site found at 772



References

[1]Ivanov, Ivan E et al. “Atomic force and super-resolution microscopy support a role for LapA as a cell-surface biofilm adhesin of Pseudomonas fluorescens.” Research in microbiology vol. 163,9-10 (2012): 685-91. doi:10.1016/j.resmic.2012.10.001