Difference between revisions of "Part:BBa K1976048"

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<partinfo>BBA_K1976048 SequenceAndFeatures</partinfo>
 
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===Characterization from Igem Team UNILausanne 2020===
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Functional kill switch assay with IPTG and aTc gradients on agar plate
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[[File:BBa K3482004 kill-switch plate.jpeg|200px|]]
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E.coli Nissle 1917 were transformed with a plasmid containing the part BBa K3482004 and the part <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K3482014"> BBa_K3482014 </a></html> (IM2 antitoxin part) and plated with a gradient of aTc and IPTG on agar plate.
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The plate shows strong activity of the IM2 antitoxin with aTc induction, whereas IPTG induction promotes production of the MiniColicin E2 toxin, resulting in a number of surviving cells (probable mutants) proportional to the dilution of the plated culture.
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We also tested our pKC1 plasmid encoding for the miniColicin E2 toxin and IM2 antitoxin in <i>E. coli Nissle 1917 ΔclbA</i>. Again, we used different concentrations of IPTG and aTc to study the effect of differential expression of the toxin and antitoxin on the growth of our strain. We compared this to the strain transformed with the empty vector pAND
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[[File:BBa K3482004 atciptg2.png|800px]]
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Dose-response growth curve of <i>E. coli Nissle 1917 ΔclbA</i> harboring kill switch plasmid pKC1 at 37°C. <i>E. coli Nissle</i> with pAND (red line), E. coli Nissle with pKC1 (green line). The lines and shade represent the mean ± standard error.
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With <i>E. coli Nissle 1917</i> with pKA1, we observed desired growth inhibition of the strain with pKC1 at high IPTG and low aTc concentrations, while the pAND strain showed no alteration in growth in any of the tested conditions. Contrastingly to <i>E. coli Nissle 1917</i> transformed with pKA1, the induction of aTc was clearly able to rescue cell growth. For increasing concentrations of IPTG, also increasing concentrations of aTc were necessary to rescue cell growth. Similar to <i>E. coli Nissle 1917</i> pKA1, cell growth could be completely inhibited for 6-8 h (IPTG: 100 µM, aTc: 0 ng/mL) before observing rapid growth. Also similarly to pKA1, the reduction in IPTG concentration gave a lesser but more constant growth inhibition, which also persisted to at least 10 h.
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Overall, we demonstrated that the miniColicin E2/IM2 encoding pKC1 plasmid could efficiently inhibit the growth of E. coli Nissle 1917 at 100 µM IPTG for up to 6 h and provided even longer growth inhibition at 5 µM IPTG.
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[1] Cascales et al, Colicin Biology, Microbiology and Molecular Biology Reviews, vol. 71, pp. 158-229, 2007<br>
 
[1] Cascales et al, Colicin Biology, Microbiology and Molecular Biology Reviews, vol. 71, pp. 158-229, 2007<br>
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[2] Wojdyla et al, Journal of Molecular Biology, vol. 417, Issues 1–2, 16 March 2012, Pages 79–94
 
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Latest revision as of 00:27, 28 October 2020

Colicin E2 DNase domain "miniColicin"

Colicin E2 is a peptide, that is toxic to coliform bacteria. It is one of a group of proteins of this type, it being a nicking endonuclease. It has a corresponding activity inhibiting protein called immunity protein Im2, which is also in the registry under the name BBa_K1976027.

Figure 1:Structure of Colicin E2 DNase domain derived from a molecular dynamics simulation.


Usage

The Colicin E2 immunity protein (BBa_K1976027 and BBa_K1976028) can be used in combination with the Colicin E2 DNase. Due to to the high binding affinity of the immunity protein to the DNase domain (Kd ∼ 10− 15 M)2, it can be used to fuse proteins and in combination with a suppression mechanism for the immunity protein as a bacterial killswitch.

Characteristics


Molecular Weight 15.2 kDa
Residues 133
pI 9.06
[Data taken created in Snapgene and http://isoelectric.ovh.org/calculate.php]
References

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 28
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization from Igem Team UNILausanne 2020

Functional kill switch assay with IPTG and aTc gradients on agar plate

BBa K3482004 kill-switch plate.jpeg

E.coli Nissle 1917 were transformed with a plasmid containing the part BBa K3482004 and the part BBa_K3482014 (IM2 antitoxin part) and plated with a gradient of aTc and IPTG on agar plate. The plate shows strong activity of the IM2 antitoxin with aTc induction, whereas IPTG induction promotes production of the MiniColicin E2 toxin, resulting in a number of surviving cells (probable mutants) proportional to the dilution of the plated culture.


We also tested our pKC1 plasmid encoding for the miniColicin E2 toxin and IM2 antitoxin in E. coli Nissle 1917 ΔclbA. Again, we used different concentrations of IPTG and aTc to study the effect of differential expression of the toxin and antitoxin on the growth of our strain. We compared this to the strain transformed with the empty vector pAND


BBa K3482004 atciptg2.png

Dose-response growth curve of E. coli Nissle 1917 ΔclbA harboring kill switch plasmid pKC1 at 37°C. E. coli Nissle with pAND (red line), E. coli Nissle with pKC1 (green line). The lines and shade represent the mean ± standard error.

With E. coli Nissle 1917 with pKA1, we observed desired growth inhibition of the strain with pKC1 at high IPTG and low aTc concentrations, while the pAND strain showed no alteration in growth in any of the tested conditions. Contrastingly to E. coli Nissle 1917 transformed with pKA1, the induction of aTc was clearly able to rescue cell growth. For increasing concentrations of IPTG, also increasing concentrations of aTc were necessary to rescue cell growth. Similar to E. coli Nissle 1917 pKA1, cell growth could be completely inhibited for 6-8 h (IPTG: 100 µM, aTc: 0 ng/mL) before observing rapid growth. Also similarly to pKA1, the reduction in IPTG concentration gave a lesser but more constant growth inhibition, which also persisted to at least 10 h. Overall, we demonstrated that the miniColicin E2/IM2 encoding pKC1 plasmid could efficiently inhibit the growth of E. coli Nissle 1917 at 100 µM IPTG for up to 6 h and provided even longer growth inhibition at 5 µM IPTG.



[1] Cascales et al, Colicin Biology, Microbiology and Molecular Biology Reviews, vol. 71, pp. 158-229, 2007
[2] Wojdyla et al, Journal of Molecular Biology, vol. 417, Issues 1–2, 16 March 2012, Pages 79–94