Difference between revisions of "Part:BBa I0500"

Revision as of 14:44, 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 Glasgow 2017] improved this part by splitting its constituent parts into two separate BioBricks: BBa_K2442101 encoding minimal pBAD and BBa_K2442104 encoding AraC under regulation of LacI-regulated promoter. This allows for greater control over arabinose-inducible systems. Placing AraC under control of LacI regulated promoter allows to induce expression of AraC only when required, taking translational load off grown cells. AraC coding sequence is also available as part BBa_K2442103, which can be placed under other promoter of choice. We isolated the minimal DNA sequence from the native pBAD that is sufficient to retain its function. The promoter retains all the operator sites O₁, O₂, I₁ and I₂ required for binding of AraC. As some of these sites lie within the PC promoter and overlap with AraC coding sequence, the minimal pBAD has the araC start codon ATG mutated to AGT to prevent the protein from being made. See part BBa_K2442101 for details. When transformed into AraC-positive E. coli strains such as DH5α, pBAD is sufficiently activated and does not require introduction of AraC from another plasmid. DH5α carrying pBAD+GFP reporter plasmid BBa_K2442102 showed 2000x fold increase in fluorescence on arabinose plate compared to control (see part BBa_K2442102 ) for details.

[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.

Revision as of 14:32, 30 October 2017 (view source) Natalka (Talk \| contribs) ← Older edit		Revision as of 14:44, 30 October 2017 (view source) Natalka (Talk \| contribs) Newer edit →
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	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 [https://parts.igem.org/Part:BBa_I0500:Experience Experience].		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 [https://parts.igem.org/Part:BBa_I0500:Experience Experience].

−	[http://2017.igem.org/Team:Glasgow Team 2017] improved this part	+	[http://2017.igem.org/Team:Glasgow Team Glasgow 2017] improved this part by splitting its constituent parts into two separate BioBricks: [https://parts.igem.org/Part:BBa_K2442101 BBa_K2442101] encoding minimal pBAD and [https://parts.igem.org/Part:BBa_K2442104 BBa_K2442104] encoding AraC under regulation of LacI-regulated promoter. This allows for greater control over arabinose-inducible systems. Placing AraC under control of LacI regulated promoter allows to induce expression of AraC only when required, taking translational load off grown cells. AraC coding sequence is also available as part [https://parts.igem.org/Part:BBa_K2442103 BBa_K2442103], which can be placed under other promoter of choice. We isolated the minimal DNA sequence from the native pBAD that is sufficient to retain its function. The promoter retains all the operator sites O<sub>1</sub>, O<sub>2</sub>, I<sub>1</sub> and I<sub>2</sub> required for binding of AraC. As some of these sites lie within the PC promoter and overlap with AraC coding sequence, the minimal pBAD has the ''araC'' start codon ATG mutated to AGT to prevent the protein from being made. See part [https://parts.igem.org/Part:BBa_K2442101 BBa_K2442101] for details. When transformed into AraC-positive ''E. coli'' strains such as DH5α, pBAD is sufficiently activated and does not require introduction of AraC from another plasmid. DH5α carrying pBAD+GFP reporter plasmid [https://parts.igem.org/Part:BBa_K2442102 BBa_K2442102] showed 2000x fold increase in fluorescence on arabinose plate compared to control (see part [https://parts.igem.org/Part:BBa_K2442102 BBa_K2442102] ) for details.