Difference between revisions of "Part:BBa K2992037"

Latest revision as of 14:47, 21 October 2019

Promoterless gusA construct

Usage and Biology

This parts entry represents a promoterless GusA reporter construct. The construct comprises the reporter gene gusA from E. coli BBa_K2300032 and the 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

The GusA reporter system was used to assess the promoter activity of our promoters chosen to drive the expression of botR from the genome of C. sporogenes and the expression of plasmid-borne acetone production pathways. The three composite parts, promoterless gusA construct BBa_K2992037, gusA reporter construct with Pfdx 5-UTR+RBS BBa_K2992038 and gusA reporter construct with P-ntnH 5-UTR+RBS BBa_K2992039 were assembled onto pMTL82121 plasmids and transformed into wild-type or promoter-botR genome integrants of C. sporogenes. GusA activity was then measured from the resultant cell lysates.

Pfdx provided considerable reporter activity in wild-type C. sporogenes (purple lines). As expected, PntnH was incapable of generating sufficient reporter without the presence of genomic botR (green lines). In the presence of botR, the construct generated detectable GusA (red lines) thus validating the positive transcriptional activation of Pntnh by the gene product of botR. Collectively these data indicate sufficient activity of PbotR and PntnH whilst demonstrating the critical activation of PbotR by the sigma factor BotR. Taken together, we conclude that the botR integration constructs should be applicable for acetone production studies. 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, J., Pennington, O., Cartman, S. and Minton, N. (2009). A modular system for Clostridium shuttle plasmids. Journal of Microbiological Methods, 78(1), pp.79-85.

Joseph, R.C., Kim, N.M. & Sandoval, N.R., 2018. Recent Developments of the Synthetic Biology Toolkit for Clostridium. Frontiers In Microbiology, 9, p.154.

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-This parts entry represents a promoterless GusA reporter construct. The construct comprises the reporter gene <i>gusA</i> from <i>E. coli [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 promoterless GusA reporter construct. 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>
 <!-- -->
 ===Characterisation===
-Data incoming.
+The GusA reporter system was used to assess the promoter activity of our promoters chosen to drive the expression of <i>botR</i> from the genome of <i>C. sporogenes</i> and the expression of plasmid-borne acetone production pathways. The three composite parts, promoterless <i>gusA</i> construct [https://parts.igem.org/Part:BBa_K2992037 BBa_K2992037], <i>gusA</i> reporter construct with P<i>fdx</i> 5-UTR+RBS
+[https://parts.igem.org/Part:BBa_K2992038 BBa_K2992038] and <i>gusA</i> reporter construct with P-<i>ntnH</i> 5-UTR+RBS [https://parts.igem.org/Part:BBa_K2992039 BBa_K2992039] were assembled onto pMTL82121 plasmids and transformed into wild-type or promoter-<i>botR</i> genome integrants of <i>C. sporogenes</i>. GusA activity was then measured from the resultant cell lysates.
+[[File:GusA botR.png]]
+<BR>P<i>fdx</i> provided considerable reporter activity in wild-type <i>C. sporogenes</i> (purple lines). As expected, P<i>ntnH</i> was incapable of generating sufficient reporter without the presence of genomic <i>botR</i> (green lines). In the presence of <i>botR</i>, the construct generated detectable GusA (red lines) thus validating the positive transcriptional activation of P<i>ntnh</i> by the gene product of <i>botR</i>. Collectively these data indicate sufficient activity of P<i>botR</i> and P<i>ntnH</i> whilst demonstrating the critical activation of P<i>botR</i> by the sigma factor BotR.  Taken together, we conclude that the <i>botR</i> integration constructs should be applicable for acetone production studies.
+<span class='h3bb'>Sequence and Features</span> <br><br>
-<span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K2992037 SequenceAndFeatures</partinfo>
 ===References===
-Heap Modular
+Heap, J., Pennington, O., Cartman, S. and Minton, N. (2009). A modular system for Clostridium shuttle plasmids. Journal of Microbiological Methods, 78(1), pp.79-85.
-Joseph Recent Developments of the Synthetic Biology Toolkit for Clostridium
+Joseph, R.C., Kim, N.M. & Sandoval, N.R., 2018. Recent Developments of the Synthetic Biology Toolkit for Clostridium. Frontiers In Microbiology, 9, p.154.