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 ([http://partsregistry.org/Part:pSB3K3 pSB3K3]) in cells with a titratable response to arabinose.

pBAD Promoter

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

• 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.

Therefore, the araC-pBAD system offers regulatable control of gene expression in the presence of the inducer and highly repressed in the absence of the inducer.

The native arabinose system is used by E.coli to:

• take up L-arabinose from the growth medium by high-capacity and low-affinity trasporters: AraE and AraFGH;
• convert intracellular arabinose into D-xylulose-5-phosphate in three reactions catalyzed by the products of the genes from the pBAD operon.

AraC protein functions as a homodimer. The monomer possesses two domain - a dimerization domain that also binds arabinose and a DNA-binding domain.

AraC repressor regulates expression of its own synthesis and the other genes of the arabinose system. In the absence of arabinose:

• AraC represses transcription initiation at the pBAD promoter. The araC dimer binding to its operator sites creates a DNA loop which sterically blocks access of RNA polymerase to the pBAD promoter.
• AraC represses expression of its own.

In the presence of arabinose:

• AraC stimulates transcription of araE and araFGH genes.
• AraC activates transcription from the pBAD promoter.
• AraC represses expression of its own.

Therefore, the linear induction with increasing arabinose concentration is not achievable in the standard E.coli strain, because the endogenous promoter which controls the araE gene is upregulated by an increasing concentration of arabinose. Thus, with higher level of the monosaccharide in the medium, more relevant transporters are present in the plasma membrane and therefore the rate of uptake rises accordingly.
This feedback regulation leads to all-or-none expression of genes under the pBAD promoter. Rather than varying the level of gene expression in individual cells of the culture, the concentration of arabinose in the medium changes the fraction of cells that are fully induced.

This induction of arabinose transporter can be circumvented by deleting the chromosomal araE gene and replacing it with a plasmid-borne copy of the araE under the control of a constitutive promoter.

One of the strains adapted for this purpose is [http://cgsc.biology.yale.edu/Strain.php?ID=111773 BW27783]], which we obtained from...

It is also important to mention that the titratable response is additionally affected by degradation of the arabinose at low concentrations of the monosaccharide. Arabinose breakdown is mediated by the araBAD genes.

Another arabinose transporter is araFGH and it should also be constitutively expressed to guarantee linear response.

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.

pBAD is an E.coli promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (BBa_I13458) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription [1]. In the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription [1].

==E.coli Strain In order to obtain a linear titratable relation between the concentration of arabinose in the medium and the level of reflectin expression, a special strain of bacteria needs to be used.

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:

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.

References

Khlebnikov, A., K.A. Datsenko, T. Skaug, B.L. Wanner, J.D. Keasling 2001. Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiol 147:3241-3247

Regulation of the l-arabinose operon of Escherichia coli Robert Schleif,

Biology Dept, Johns Hopkins University, 3400 N. Charles St, Baltimore, MD 21218, USA