Difference between revisions of "Part:BBa K4604024:Design"
 
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===Design Notes===
 
===Design Notes===
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This was set up to test the toxicity of MazF without the regulation of the AdoCbl riboswitch used in BBa_K4604018.
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This was set up to test the toxicity of MazF without the regulation of the AdoCbl riboswitch used in <a href="https://parts.igem.org/Part:BBa_K4604018">BBa_K4604018</a>.</html> The naturally occurring toxin/antitoxin system is also present in the strain we are using. Because of this, a constitutive level of toxin/antitoxin expression is given. The endogenous toxin-antitoxin complex regulates its own gene expression [1], thus we decided to use a different promoter than the native one to prevent interference by the toxin/antitoxin complex.
  
  
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===Cloning of piG_23a===
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===Cloning of piG_23===
  
 
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A plasmid given to us from our PI was used as a backbone and amplified according to the protocol for the Q5 polymerase with an annealing temperature 55°C and elongation time of 3 minutes. For the PCR of the insert (<i>mazF</i> expressional unit) we also used the general protocol for the Q5 polymerase with an annealing temperature of 58°C and an elongation time of 4 minutes. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Gibsson Assembly was used according to the protocol to assemble the plasmid. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.  
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  A plasmid given to us from our PI was used as a backbone and amplified according to the protocol for the Q5 polymerase with an annealing temperature 55°C and elongation time of 3 minutes. For the PCR of the insert (<i>mazF</i> expressional unit) we also used the general protocol for the Q5 polymerase with an annealing temperature of 58°C and an elongation time of 4 minutes. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Gibson Assembly was used according to the protocol to assemble the plasmid. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.  
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===Source===
 
===Source===
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Parts of BBa_K4604018 were inserted into a backbone from our PI with Gibson Assembly.
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Parts of <a href="https://parts.igem.org/Part:BBa_K4604018">BBa_K4604018</a> were inserted into a backbone from our PI with Gibson Assembly.
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===References===
 
===References===
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[1] Marianovsky I, Aizenman E, Engelberg‐Kulka H, Glaser G. The Regulation of the Escherichia coli mazEF Promoter Involves an Unusual Alternating Palindrome. Journal of Biological Chemistry [Internet]. 2001 Feb 1;276(8):5975–84. Available from: https://doi.org/10.1074/jbc.m008832200

Latest revision as of 19:05, 11 October 2023


piG_23 (tetR_mazF)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 701
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 701
    Illegal BglII site found at 710
    Illegal BamHI site found at 1105
    Illegal XhoI site found at 1114
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 701
    Illegal XbaI site found at 616
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This was set up to test the toxicity of MazF without the regulation of the AdoCbl riboswitch used in BBa_K4604018. The naturally occurring toxin/antitoxin system is also present in the strain we are using. Because of this, a constitutive level of toxin/antitoxin expression is given. The endogenous toxin-antitoxin complex regulates its own gene expression [1], thus we decided to use a different promoter than the native one to prevent interference by the toxin/antitoxin complex.


Cloning of piG_23

A plasmid given to us from our PI was used as a backbone and amplified according to the protocol for the Q5 polymerase with an annealing temperature 55°C and elongation time of 3 minutes. For the PCR of the insert (mazF expressional unit) we also used the general protocol for the Q5 polymerase with an annealing temperature of 58°C and an elongation time of 4 minutes. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Gibsson Assembly was used according to the protocol to assemble the plasmid. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.

Source

Parts of BBa_K4604018 were inserted into a backbone from our PI with Gibson Assembly.

References

[1] Marianovsky I, Aizenman E, Engelberg‐Kulka H, Glaser G. The Regulation of the Escherichia coli mazEF Promoter Involves an Unusual Alternating Palindrome. Journal of Biological Chemistry [Internet]. 2001 Feb 1;276(8):5975–84. Available from: https://doi.org/10.1074/jbc.m008832200