Low Level Expression

For our in vivo work, we needed to be able to express reflectin at low levels, and control the level of expression reliably. Therefore, we expressed reflectin under an arabinose inducible promoter (pBad) on a low copy plasmid (PSB3K3) in cells with a titratable response to arabinose.

Constructs

In this experiment we used confocal microscopy to compare distribution of Reflectin A1 when it is expressed at high and low levels in E.coli cells. In order to do so, we used four different plasmids with Reflectin A1 gene expressed under the control of the pBAD promoter, and their assembly is described in this section. The constructs we relied on are the following:

Strains of E.coli

To obtain a linear titratable relation between the concentration of arabinose in the medium and the level of the gene reflectin expression under the control of the pBAD promoter, a special strain of bacteria needs to be used.

The inducible pBAD promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_I0500 I0500] we used is tightly controlled by two factors:

• [http://en.wikipedia.org/wiki/Arabinose L-arabinose] monosaccharide taken up by the cell from the medium, which acts as an inducer.
• AraC protein included in the I0500 biobrick, which acts an a repressor.

Inducible pBad/araC promoter pBad is an E. coli promoter that is tightly controlled by: inducer: L-arabinose. repressor: AraC apparently acts as the repressor one study concluded that 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.))

Team Slovenia 2010 further characterized pBAD promoter. Check results on Experience.

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.

Observations

When reflectin was expressed on a low copy plasmid, we saw fewer inclusion bodies than when expressed on a high copy plasmid.

Induction

Using a plate reader, we measured the expression of reflectin-GFP over time after inducing with arabinose. We saw that reflectin does not appear to be particularly toxic to E. Coli.