Difference between revisions of "Part:BBa K4252023"

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<partinfo>BBa_K4252023 parameters</partinfo>
 
<partinfo>BBa_K4252023 parameters</partinfo>
 
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==Characterization==
 +
===Expression Test===
 +
====Aim====
 +
To verify that the temperature sensitive regulating element is controlled by temperature.
 +
 +
====Method====
 +
*'''Induction''':
 +
Plasmid construction：A plasmid containing temperature sensitive regulatory element cI857, R promoter and red fluorescent protein mCherry<br />
 +
*'''Plasmid construction''':
 +
Use snapgene to edit and synthesize cI857 and R promoter in the company, and use homologous recombination to introduce red fluorescent protein into plasmid.<br />
 +
====Results====
 +
The sequencing result is no problem. cI857, R promoter and mCherry are all in the plasmid.
  
 
===Function Test===
 
===Function Test===
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From the fluorescence data corresponding to our temperature gradient, the red fluorescence expression was very weak in the temperature range of 30 ℃ to 34 ℃, indicating that the dimer formed by cI857 inhibited the R promoter at this time, leading to the subsequent weakening of mCherry expression; The strong red fluorescence expression at 37 ℃ to 42 ℃ indicated that cI857 dimer depolymerized and the inhibition of R promoter was relieved, and then mCherry was normally expressed. <br />
 
From the fluorescence data corresponding to our temperature gradient, the red fluorescence expression was very weak in the temperature range of 30 ℃ to 34 ℃, indicating that the dimer formed by cI857 inhibited the R promoter at this time, leading to the subsequent weakening of mCherry expression; The strong red fluorescence expression at 37 ℃ to 42 ℃ indicated that cI857 dimer depolymerized and the inhibition of R promoter was relieved, and then mCherry was normally expressed. <br />
 
In order to determine the better culture time, we selected the values of 12h and 15h for comparison.We found that there was little difference between the absorbance of 12h culture and 15h culture, and even 15h would decrease under some temperatures, so we chose the final culture time of 12h.<br />
 
In order to determine the better culture time, we selected the values of 12h and 15h for comparison.We found that there was little difference between the absorbance of 12h culture and 15h culture, and even 15h would decrease under some temperatures, so we chose the final culture time of 12h.<br />
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[[File:mCherry figure 1.png|500px|thumb|centre|Figure 1]]
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[[File:mCherry figure 2.png|500px|thumb|centre|Figure 2]][[File:mCherry figure 1.png|500px|thumb|centre|Figure 1]]
 
<br />
 
<br />
  

Revision as of 16:35, 11 October 2022


cI857-R promoter-phlF-pst plasmid

cI857-R promoter-phlF-pst plasmid involved: The temperature-sensitive control element cI857(BBa_K200011) R promoter, phlF repressor(BBa_K1725040) pst(PstS:BBa_K4252005;PstC BBa_K4252006;PstA: BBa_K4252007;PstB: BBa_K4252008;PstSCAB:BBa_K4252009). We use different temperatures to culture the strain. It is estimated that cI857 forms dimer under 30℃ culture conditions to inhibit the R promoter, so that its downstream phlf and pst do not express. The cI857 dimer was depolymerized at 37 ℃, and the inhibition of the R promoter was relieved. The downstream phlf and pst were normally expressed.

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 AgeI site found at 1685
    Illegal AgeI site found at 2251
    Illegal AgeI site found at 4166
    Illegal AgeI site found at 5180
    Illegal AgeI site found at 5369
    Illegal AgeI site found at 5935
    Illegal AgeI site found at 7936
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 5000
    Illegal SapI.rc site found at 8952


Characterization

Expression Test

Aim

To verify that the temperature sensitive regulating element is controlled by temperature.

Method

  • Induction:

Plasmid construction：A plasmid containing temperature sensitive regulatory element cI857, R promoter and red fluorescent protein mCherry

  • Plasmid construction:

Use snapgene to edit and synthesize cI857 and R promoter in the company, and use homologous recombination to introduce red fluorescent protein into plasmid.

Results

The sequencing result is no problem. cI857, R promoter and mCherry are all in the plasmid.

Function Test

Aim

we cultured the plasmids transferred into cI857,phlF,R promoter and mCherry at 30℃,33℃,37℃ and 42℃,in order to verify that the temperature sensitive regulating element is controlled by temperature.

Method

  1. First,transfer the plasmid into BL21 strain.
  2. The strain transferred into plasmid was screened for resistance to ensure successful transfer. The method used in this step is monoclonal antibody.
  3. Pick out the strains growing on the resistant culture dish and cultivate them at 30 ℃, 33 ℃, 37 ℃ and 42 ℃ for 12h and 15h.
  4. Absorbance test of cultured bacteria.(OD600)
  5. Crush the cultured bacteria to prevent their continued expression.
  6. Fluorescence test of the broken bacteria under the microplate reader.
  7. Calculate the ratio of fluorescence intensity to absorbance and record it as the relative fluorescence intensity.
  8. Plot with temperature and relative fluorescence intensity.

Results

From the fluorescence data corresponding to our temperature gradient, the red fluorescence expression was very weak in the temperature range of 30 ℃ to 34 ℃, indicating that the dimer formed by cI857 inhibited the R promoter at this time, leading to the subsequent weakening of mCherry expression; The strong red fluorescence expression at 37 ℃ to 42 ℃ indicated that cI857 dimer depolymerized and the inhibition of R promoter was relieved, and then mCherry was normally expressed.
In order to determine the better culture time, we selected the values of 12h and 15h for comparison.We found that there was little difference between the absorbance of 12h culture and 15h culture, and even 15h would decrease under some temperatures, so we chose the final culture time of 12h.

Figure 2
Figure 1


In this part, we cultured the plasmids transferred into cI857, phlF, R promoter and mCherry at 30℃ ,33℃ ,37℃ and 42℃ . From the fluorescence data corresponding to our temperature gradient, the red fluorescence expression was very weak in the temperature range of 30 ℃ to 34 ℃, indicating that the dimer formed by cI857 inhibited the R promoter at this time, leading to the subsequent weakening of mCherry expression; The strong red fluorescence expression at 37 ℃ to 42 ℃ indicated that cI857 dimer depolymerized and the inhibition of R promoter was relieved, and then mCherry was normally expressed.
This part has verified that the temperature sensitive control element cI857 is controlled by temperature.

References

Wang X, Han JN, Zhang X, Ma YY, Lin Y, Wang H, Li DJ, Zheng TR, Wu FQ, Ye JW, Chen GQ. Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun. 2021 Mar 3;12(1):1411. doi: 10.1038/s41467-021-21654-x. PMID: 33658500; PMCID: PMC7930084.