Difference between revisions of "Part:BBa K2992012"

Revision as of 01:20, 22 October 2019

PbotR from C. botulinum

Promoter region for botR in C. botulinum

Usage and Biology

This promoter region is found naturally upstream of the botR 5’-UTR in C. botulinum BBa_K2992014. BotR is an alternative sigma factor involved in the positive regulation of botulinum neurotoxin and associated genes. In our project, we use PbotR to drive the expression of botR (BBa_K2992002) which in turn, regulates the production of our reporter genes which we have placed under the control of a BotR-activated promoter (BBa_K2992028, BBa_K2992029, BBa_K2992030, BBa_K2992034, BBa_K2992035, BBa_K2992036). Doing so allowed us to link BotR expression with the trasncriptional activation of our reporter genes.

Characterisation

This basic part was used for the assembly of our composite parts and characterised using using FAST and acetone assays. More information can be found on our Results Page.

Characterisation of this promoter against Pfdx, Pthl and Pntnh using FAST fluorescent assay, showed PbotR to be a relatively weak promoter in both E.coli and and C.sporogenes.

The data demonstrated acetone production of >2mM when using PbotR promoter or PpyrKDE to drive expression of botR in the chromosome of C. sporogenes. Considerable acetone production (4-6 mM) was observed when using the constitutive clostridial promoter Pfdx, driving directly the expression of the acetone operon genes. Crucially, acetone production was almost undetectable when botR was absent from the genome of C. sporogenes and when no promoter was used to drive expression of the acetone production operon. These data provide experimental validation for the production of acetone in C. sporogenes as a model for botulinum toxin prediction in foodstuffs.

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

Raffestin, S., Dupuy, B., Marvaud, J. and Popoff, M. (2004). BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. Molecular Microbiology, 55(1), pp.235-249.

Zhang, Z., Korkeala, H., Dahlsten, E., Sahala, E., Heap, J., Minton, N. and Lindström, M. (2013). Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502. PLoS Pathogens, 9(3), p.e1003252.

@@ Line 17: / Line 17: @@
 https://2019.igem.org/wiki/images/6/6b/T--Nottingham--Basic3.png
 <br>
-Characterisation of this promoter against P<i>fdx</i>, P<i>thl</i> and P<i>ntnh</i> using FAST fluorescent assay, showed P<i>BotR</i> to be a mild promoter in E.<i>coli</i> and only slightly stronger than no promoter in C.<i>sporogenes</i>. <br> In the C. <i>sporogenes</i> experiments, adequate expression was detected for each of the clostridial promoters chosen for study. The two Pfdx derivatives generated the greatest level of reporter activity whilst the two C. <i>botulinum</i> promoters generated much lower levels of activity. Reporter activity appeared to be generally higher when analysed from the E. <i>coli</i> lysates as opposed to the C. <i>sporogenes</i> lysates. In those experiments, activity from the P<i>botR</i> and P<i>ntnh</i> constructs were considerably greater than the no promoter control. <br>
+Characterisation of this promoter against P<i>fdx</i>, P<i>thl</i> and P<i>ntnh</i> using FAST fluorescent assay, showed P<i>botR</i> to be a relatively weak promoter in both E.<i>coli</i> and and C.<i>sporogenes</i>. <br>
 [[File:Acetone data.png]]
 <br>
-The data demonstrated appreciable acetone production of >2nM concentration when using either the native P<i>botR</i> promoter and associated 5’-UTR+RBS or the RBS only construct to permit polar transcription from P<i>pyrKDE</i>. Considerable acetone production (4-6nM) was observed when using the constitutive clostridial promoter P<i>fdx</i>. Crucially, acetone production was comparably scant when <i>botR</i> was absent from the genome of <i>C. sporogenes</i> and when no promoter was used to drive expression of the acetone production operon. These data provide experimental validation for the production of acetone in <i>C. sporogenes</i> as a model for Botulinum toxin prediction in foodstuffs.
+The data demonstrated acetone production of >2mM when using P<i>botR</i> promoter or P<i>pyrKDE</i> to drive expression of <i>botR</i> in the chromosome of <i>C. sporogenes</i>. Considerable acetone production (4-6 mM) was observed when using the constitutive clostridial promoter P<i>fdx</i>, driving directly the expression of the acetone operon genes. Crucially, acetone production was almost undetectable when <i>botR</i> was absent from the genome of <i>C. sporogenes</i> and when no promoter was used to drive expression of the acetone production operon. These data provide experimental validation for the production of acetone in <i>C. sporogenes</i> as a model for botulinum toxin prediction in foodstuffs.