Difference between revisions of "Part:BBa K2992039"
(→‎Usage and Biology)
Line 6: Line 6:
  
 
===Usage and Biology===
 
===Usage and Biology===
−
This parts entry represents a <i>gusA</i> reporter construct under the regulatory control of promoter for the non-toxic non-haemagglutinin <i>ntnH</i> gene of <i>C. botulinum</i> [https://parts.igem.org/Part:BBa_K2992001 BBa_K2992001] and associated 5’UTR+RBS [https://parts.igem.org/Part:BBa_K2992015 BBa_K2992015]. The construct comprises the reporter gene <i>gusA</i> from <i>E. coli</i> [https://parts.igem.org/Part:BBa_K2300032 BBa_K2300032] and the strong clostridial terminator T<i>Fad</i> from <i>C. pasteurianum</i>  [https://parts.igem.org/Part:BBa_K2284012 BBa_K2284012]. <i>gusA</i> encodes a B-glucorinidase from <i>E. coli</i> which can be used in a histochemical reaction to quantitatively measure promoter strength. This is accomplished through the addition of the fluorescent substrate 4 methylumbelliferyl glucuronide (4-MUG) which undergoes a colorimetric reaction with GusA, proportionate to its intracellular concentration. The nature of the Gus assay permits the measurement of promoter strength directly from cell debris without the requirement for extended cell lysis procedures. Doing so allows for the detection of reporter gene-products from anaerobic organisms which have recently been removed from the anaerobic cabinet or which have been frozen directly thereafter. In our project we use <i>gusA</i> as an established reporter gene for anaerobic organisms to validate the promoters chosen for our <i>botR</i> and acetone production constructs.  <br><br>
+
This parts entry represents a <i>gusA</i> reporter construct under the regulatory control of promoter for the non-toxic non-haemagglutinin <i>ntnH</i> gene of <i>C. botulinum</i>. The construct comprises P<i>ntnH</i> [https://parts.igem.org/Part:BBa_K2992001 BBa_K2992001] and its associated 5’-UTR and RBS driving the expression of the reporter gene <i>gusA</i> from <i>E. coli</i> [https://parts.igem.org/Part:BBa_K2300032 BBa_K2300032] with a strong clostridial terminator T<i>Fad</i> from <i>C. pasteurianum</i>  [https://parts.igem.org/Part:BBa_K2284012 BBa_K2284012]. <i>gusA</i> encodes a B-glucorinidase from <i>E. coli</i> which can be used in a histochemical reaction to quantitatively measure promoter strength. This is accomplished through the addition of the fluorescent substrate 4 methylumbelliferyl glucuronide (4-MUG) which undergoes a colorimetric reaction with GusA, proportionate to its intracellular concentration. The nature of the Gus assay permits the measurement of promoter strength directly from cell debris without the requirement for extended cell lysis procedures. Doing so allows for the detection of reporter gene-products from anaerobic organisms which have recently been removed from the anaerobic cabinet or which have been frozen directly thereafter. In our project we use <i>gusA</i> as an established reporter gene for anaerobic organisms to validate the promoters chosen for our <i>botR</i> and acetone production constructs.  <br><br>
  
 
<!-- -->
 
<!-- -->

Revision as of 15:12, 30 September 2019

gusA reporter construct with PntnH 5-UTR+RBS



Usage and Biology

This parts entry represents a gusA reporter construct under the regulatory control of promoter for the non-toxic non-haemagglutinin ntnH gene of C. botulinum. The construct comprises PntnH BBa_K2992001 and its associated 5’-UTR and RBS driving the expression of the reporter gene gusA from E. coli BBa_K2300032 with a strong clostridial terminator TFad from C. pasteurianum BBa_K2284012. gusA encodes a B-glucorinidase from E. coli which can be used in a histochemical reaction to quantitatively measure promoter strength. This is accomplished through the addition of the fluorescent substrate 4 methylumbelliferyl glucuronide (4-MUG) which undergoes a colorimetric reaction with GusA, proportionate to its intracellular concentration. The nature of the Gus assay permits the measurement of promoter strength directly from cell debris without the requirement for extended cell lysis procedures. Doing so allows for the detection of reporter gene-products from anaerobic organisms which have recently been removed from the anaerobic cabinet or which have been frozen directly thereafter. In our project we use gusA as an established reporter gene for anaerobic organisms to validate the promoters chosen for our botR and acetone production constructs.


Characterisation

Data incoming.

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]

References

Heap Modular Joseph Recent Developments of the Synthetic Biology Toolkit for Clostridium