Part:BBa_K316008
Cleavable GFP-XylE fusion with Pveg promoter and terminator
This part contains BBa_K316007 with added double terminator BBa_B0014. This construct 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. Terminator BBa_B0014 has been added to comply with Biobrick standards. This particular terminator is stronger and is different from BBa_B0015.
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 Material Safety Data Sheet[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, linkers and double terminator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1229
Illegal NgoMIV site found at 1401
Illegal AgeI site found at 1752 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 785
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 (BBa_K316007) 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. Top: Constitutive XylE expression BBa_K316004, Bottom: 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.
Although the activity is leaky and small amounts of the colored product are produced, the difference is clear between two constructs. Both XylE only and GFP-XylE constructs are under Pveg promoter in the same E. coli strain, so that we can assume equal production of protein product.
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. These are described on our wiki[http://2010.igem.org/Team:Imperial_College_London/Results] and the aforementioned parts pages.
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