Difference between revisions of "Part:BBa K316007"

Latest revision as of 20:54, 5 November 2010

Recombinant GFP-XylE protein under Pveg promoter

This construct contains BBa_K316006 under the control of Pveg promoter BBa_K143053. It is designed so that the XylE activity is substantially reduced untill such a time when a TEV protease is added to the system and transcribed. TEV protease cleavable linker is positioned between the two proteins. Once the linker is cleaved, XylE is free to tetramerise and assume full activity. GFP is His tagged at the 5' end to facilitate purificaiton for in-vitro assays. This construct does not have a terminator at the end, another construct BBa_K316008 contains a double terminator BBa_B0014.

For more information about XylE, it's substrate and spectrophotometric assays, please see BBa_K316003 or our [http://2010.igem.org/Team:Imperial_College_London/Results wiki results section]

Safety

The substrate XylE works on is a chemical called catechol. It is classed as irritant in the EU but as toxic in the USA, as well as being a possible carcinogen. It should therefore be handled with care and proper safety equipment. More information is available on the [http://www.sciencelab.com/msds.php?msdsId=9927131 Material Safety Data Sheet].

Structure and Features

Figure I. Graphical representation of the GFP-XylE construct with associated Pveg promoter, tags and linkers.

Sequence and Features

Part Characterisation

To investigate whether the GFP-XylE construct does indeed work as intended and produces a response when induced, an assay was performed where catechol was added to GFP-XylE transformed E.coli. The progress of the reaction was monitored by measuring production of the yellow product, 2-hydroxymuconic semialdehyde (HMS), which arbsorbs light at 380nm, see BBa_K316003 for details. The product concentraton correlates well to the velocity of the reaction, if divided by time, and by using various initial catechol concentrations as a substrate, a kinetic profile of the reaction could be constructed.

Figure II. Production of yellow product (HMS) over time. Left: Constitutive XylE expression BBa_K316004, Right: GFP-XylE expressing cells BBa_K316007.

The results show that the N-terminal fusion of GFP gene to the XylE gene strongly affects activity of the wild type XylE. At 3 minutes after the start, XylE expressing cells saturate the system with the colored product. Therefore we can see a 10-fold reduction in rates of product production between XylE BBa_K316004 and GFP-XylE BBa_K316007 constructs.

GFP-XylE expression in the presence or absence of TEV protease

The graph presents the data acquired in an experiment to compare HMS production of cell cultures that:

1)Express XylE gene (blue)

2)Express GFP-XylE in the absence of TEV (red)

3)Express GFP-XylE along with TEV protease (green)

The results show that the GFP-XylE fusion is far less able to tetramerise to form active XylE enzymes and break down catechol. TEV protease (expressed in the cell with the GFP-XylE) cleaves the GFP-XylE, producing active XylE protein. This can be seen by comparing the blue (XylE) and the green (GFP-XylE in presence of TEV) curve data, as they show similar rates of production of HMS colored product.

The plateauing effect is caused by depletion of the catechol substrate and differences in the height of the absorbance plateaus value is due to slightly different initial concentrations of catechol substrate (both approximately 0.2mM).

Characterisation data was obtained for XylE BBa_K316003. In addition constructs under two different promoters: J23101-XylE BBa_K316004 from E. coli was used to categorise B. subtilis derived Pveg-XylE BBa_K316005. Also GFP-XylE constructs BBa_K316007 were tested to determine the effectiveness of inhibition of XylE activity by attachment of GFP and ability of TEV to cut the resulting fusion protein. These are described on our [http://2010.igem.org/Team:Imperial_College_London/Results wiki] and the aforementioned parts pages.