Difference between revisions of "Part:BBa K2992020"

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===Usage and Biology===
 
===Usage and Biology===
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The BgaR-BgaL system of <i>C. perfringens</i>  comprises the transcriptional regulator BgaR belonging to the AraC-family and the β-galactosidase BgaL which are transcribed in a regulated fashion from the divergent P<i>gaR</i> -P<i>gaL</i>  promoter. The BgaR-BgaL system regulates the expression of carbohydrate metabolic genes in response to lactose concentrations (Hartman and Melville 2011). This parts entry represents the promoter region predicted to regulate <i>bgaR</i>. Our group has recently utlised the BgaRL regulatory system in order to generate a tightly regulate inducible system for CRISPR-Cas mutagenesis in the genus <i>Clostridium</i> (Cañadas et al., 2019). In our project, we use the P<i>gaR</i> -P<i>gaL</i> regulatory system comprised of the divergent promoter and associated 5’-UTRs in conjunction with their cognate transcriptional regulator <i>bgaR</i>  (hyperlinks and descriptions) to drive the expression of our volatile and FAST reporter genes in an inducible fashion. Doing so helps us fulfil our goal of generating reporter strains for the prediction of botulinum neurotoxin production following food manufacture. <rb><br>
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The BgaR-BgaL system of <i>C. perfringens</i>  comprises the transcriptional regulator BgaR belonging to the AraC-family and the β-galactosidase BgaL which are transcribed in a regulated fashion from the divergent P<i>gaR</i> -P<i>gaL</i>  promoter. The BgaR-BgaL system regulates the expression of carbohydrate metabolic genes in response to lactose concentrations (Hartman and Melville 2011). This part entry represents the promoter region predicted to regulate <i>bgaR</i>. In our project, we use the Plac system, comprised of the divergent P<i>bgaR</i>-P<i>bgaL</i> promoter and associated 5’-UTRs in conjunction with their cognate transcriptional regulator <i>bgaR</i>. In our project, the Plac system was used to drive lactose-dependent expression of <i>botR</i>  ([https://parts.igem.org/Part:BBa_K2992002 BBa_K2992002]) which in turn, regulates the production of our reporter genes which we have placed under the control of a BotR-activated promoter ([https://parts.igem.org/Part:BBa_K2992028 BBa_K2992028], [https://parts.igem.org/Part:BBa_K2992029 BBa_K2992029], [https://parts.igem.org/Part:BBa_K2992036 BBa_K2992036]).
  
 
===Characterisation===
 
===Characterisation===

Revision as of 03:44, 22 October 2019

PbgaR component of PbgaR -PbgaL from C. perfringens

Component of the divergent PbgaR -PbgaL promoter predicted to regulate bgaR in C. perfringens.


Usage and Biology

The BgaR-BgaL system of C. perfringens comprises the transcriptional regulator BgaR belonging to the AraC-family and the β-galactosidase BgaL which are transcribed in a regulated fashion from the divergent PgaR -PgaL promoter. The BgaR-BgaL system regulates the expression of carbohydrate metabolic genes in response to lactose concentrations (Hartman and Melville 2011). This part entry represents the promoter region predicted to regulate bgaR. In our project, we use the Plac system, comprised of the divergent PbgaR-PbgaL promoter and associated 5’-UTRs in conjunction with their cognate transcriptional regulator bgaR. In our project, the Plac system was used to drive lactose-dependent 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_K2992036).

Characterisation

The Plac system is an inducible promoter system utilizing the lactose operon from C. Perfringens. This allowed us to test the maximum and minimum reporter expression range. See our results page for more information.

Plac_diagram.png

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

Cañadas, I., Groothuis, D., Zygouropoulou, M., Rodrigues, R. and Minton, N. (2019). RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. ACS Synthetic Biology, 8(6), pp.1379-1390.

Minton, N., Ehsaan, M., Humphreys, C., Little, G., Baker, J., Henstra, A., Liew, F., Kelly, M., Sheng, L., Schwarz, K. and Zhang, Y. (2016). A roadmap for gene system development in Clostridium. Anaerobe, 41, pp.104-112. 2019 RiboCas - update

Hartman, A., Liu, H. and Melville, S. (2010). Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression inClostridium perfringens. Applied and Environmental Microbiology, 77(2), pp.471-478.


Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research, 31(13), pp.3406-3415.