Difference between revisions of "Part:BBa I0500"

Revision as of 14:32, 30 October 2017

Inducible pBad/araC promoter

pBad is an E. coli promoter that is tightly controlled by:

• inducer: L-[http://openwetware.org/wiki/Arabinose arabinose].
• repressor: AraC acts as the repressor

one [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=7768852 study] concluded that [http://openwetware.org/wiki/Arabinose arabinose] can change the conformation of araC and prevent it from successfully binding to and repressing pBad.

Usage and Biology

• When grown with 0.2% arabinose, promoter is weak-medium. [jb, 5/24/04] Part may not be compatible with MC4100 as cell line is araD 139.
• MC4100 is not a good chassis for operating BBa_I0500 (pBad promoter). The feed-forward regulation of the endogenous promoter controlling expression of the arabinose transporter prevents linear induction with increasing arabinose concentration. ((Engineered strain from Keasling's lab, used by jrk for operation of the screening plasmid.))

[http://2010.igem.org/Team:Slovenia Team Slovenia 2010] further characterized pBAD promoter. Check results on Experience.

[http://2016.igem.org/Team:IISc_Bangalore IISc Bangalore 2016] showed catabolite repression of expression from the promoter by glucose. Check results on Experience.

[http://2016.igem.org/Team:OUC-China OUC-China 2016] characterized BBa_I0500 of different concentrations of L-arabinose on the transcriptional level. Check results on Experience.

[http://2017.igem.org/Team:TU-Eindhoven TU-Eindhoven 2017] optimized the expression on by testing 3 different temperatures, where 37 degrees Celcius proved most successful. Addition of L-arabinose has also been optimized to compensate for the breakdown of L-arabinose by the E.coli strain BL21(DE3) for mCherry and GFP expression. Check results on Experience.

[http://2017.igem.org/Team:Glasgow Team 2017] improved this part

• [http://openwetware.org/wiki/Titratable_control_of_pBAD_and_lac_promoters_in_individual_E._coli_cells#pBAD_promotersOpenWetWare From an OWW article on pBAD and lac promoters]:
  • Import of arabinose into cells is mediated by the araE gene. Induction of the arabinose transporter encoded by araE can be uncoupled from the endogenous PBAD promoter by deleting the chromosomal araE gene and replacing it with a plasmid-borne copy of araE under control of a constitutive promoter (1). However, this does not seem to be enough to allow for homogenous expression from PBAD promoters in a population of cells (2).
  • At low concentrations of arabinose, degradation of the sugar within cells also effects the homogeneity of expression from PBAD promoters (2). Arabinose degradation is mediated by the araBAD genes. Strains lacking functional araE, araFGH (another transporter), and araBAD can be made to be responsive to arabinose for PBAD promoter induction (2). This is achieved by introduction of a mutant lacY gene. LacY A177C allows for downhill transport of arabinose, as well as maltose, palatinose, sucrose, and cellobiose (3), but does not actively transport these sugars (4). Lactose import is not affected in this mutant. So, PBAD promoters in cells lacking endogeneous arabinose importers and containing LacY A177C are linearly responsible to arabinose at the individual cell level.
  • By the way, AraC is the repressor of the PBAD promoter. It is encoded on the pBAD vector series and is still present in the above-described strains.

PC and AraC are located on the complementary strand, reading right to left as written.

• At least one registry stock contains a deletion of the C at base 1194. This is after the transcriptional start but before the translation start, so it may not be significant. Parts with this mutation have been qualitatively observed to function normally.