Difference between revisions of "Part:BBa K4239009"
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<h2>characterization</h2>
 
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<p>To check that the synthesized DNA contains the genes <i>fiatluxCD</i> and the promotor, we carry out a miniprep of the plasmid pACYDuet-1-J23117-<i>fiatluxCD</i>. This last one is digested by two enzymes, EcoRI and PstI (iGEM restriction sites), and a migration on agarose gel was realised. The expected fragment are :  
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<p>To check that the synthesized DNA contains the <I>hok/sok</i> toxin/antitoxin system, we carry out a miniprep of the plasmid pSB1C3-<I>hok/sok</i>. This last one is digested by two enzymes, EcoRI and PstI (iGEM restriction sites), and a migration on agarose gel was realized. The expected fragments are :  
  
 
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<p>The strip at 635 bp indicates that <i>hok/sok</i> in the plasmid.</p>
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<p>Column 1: Non digested plasmid. Two strips at 2000 bp and 2600 bp indicates the supercoiled and normal form of the complete non digested plasmid. </p>
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<p>Column 2: Double digested plasmid by EcoRI and PstI. The 2 strips at 635 bp and at 2000 bp were expected, and indicate that <i>hok/sok</I> is in the plasmid.</p>
  
 
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Revision as of 14:39, 12 October 2022


toxin/antitoxin gene hok/sok


Description

Hok/sok is a toxin/antitoxin system made to be used on the same plasmid than an interest gene (the fiatlux operon, in our project). This method of selection ensures that all the bacteria present on the plant have the plasmid, and can hence emit light. Toxins have a longer life span in cells than antitoxins, so if the bacteria lose their plasmid, the toxin is no longer destroyed by the antitoxin. This results in the death of the bacteria, thus assuring the death of all fiatlux non-carriers. As a consequence, by involving a selective pressure, the toxin/antitoxin system increases the stability of the plasmid in the bacteria.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Construction

The gene hok/sok was adapted to fit the biobrick format

characterization

To check that the synthesized DNA contains the hok/sok toxin/antitoxin system, we carry out a miniprep of the plasmid pSB1C3-hok/sok. This last one is digested by two enzymes, EcoRI and PstI (iGEM restriction sites), and a migration on agarose gel was realized. The expected fragments are :

  • Non Digested plasmid : 2664 bp
  • Double Digested plasmid : 635 bp + 2030 bp

Column 1: Non digested plasmid. Two strips at 2000 bp and 2600 bp indicates the supercoiled and normal form of the complete non digested plasmid.

Column 2: Double digested plasmid by EcoRI and PstI. The 2 strips at 635 bp and at 2000 bp were expected, and indicate that hok/sok is in the plasmid.

parts7
Figure 1: Digestion and migration on agarose gel of pSB1C3-hok/sok

References

Gregor C, Gwosch KC, Sahl SJ, Hell SW. Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging. Proc Natl Acad Sci U S A. 2018 Jan 30;115(5):962-967. doi: 10.1073/pnas.1715946115. Epub 2018 Jan 16. PMID: 29339494; PMCID: PMC5798359.