Part:BBa_K316009
LacI inducible Fast Response Module, using cleavable XylE
5' his tagged GFP-TEV linker-XylE construct is pre-made in the cell under constitutive promoter. Both Pveg promoter and spoVG RBS are best suited for maximal expression in B.subtilis. While GFP is attached to the XylE monomer via the TEV cleavable linker, the catalytic activity is low. Transcription of TEV protease allows cleavage of the linker between GFP and XylE, thus XylE is free to tetramerise into a fully functional enzyme. XylE is then able to act as described in BBa_k316004
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 final construct, containing 3' strand coding, LacI inducible TEV protease (BBa_k316012) and 5' coding constitutively expressed GFP-XylE fusion protein BBa_k316006.
Please visit our wiki for complete strategy and detailed information [http://2010.igem.org/Team:Imperial_College_London].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 869
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2111
Illegal NgoMIV site found at 2283
Illegal AgeI site found at 32
Illegal AgeI site found at 2634 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1667
Please Note, BBa_k316012 is coding on the 3' strand in the opposite direction to the arrow shown, as per Figure I
Part Characterisation
Output Amplification Model
This model was mainly developed in order to determine whether simple production is better than 1-, 2- or 3-step amplification. Furthermore, an estimation of the speed of the response was desirable.
We have modelled this system to see if 2 step amplification step, by using TEV protease to cleave a GFP-XylE fusion is faster than direct translation of XylE such as one of our characterisation constructs like BBa_k316004 and BBa_k316005. Detailed results can be found on our wiki[http://2010.igem.org/Team:Imperial_College_London/Modelling]
It was shown that amplified systems easily outperform the simple production system (control)
It was concluded that there is no advantage of 3-step amplification over 2-step amplification. Therefore, the design of a 3-step amplifier was abandoned.
The results concerning the 2-step amplification module were not conclusive. It could not be firmly decided whether 2-step amplification is going to perform better than 1-step amplification. This is because several of the parameters that 2- and 1-step amplifiers are sensitive to could not be determined with certainty. 2 parameters have been recognised as crucial and decisive. Those are protein production rates and catalytic constants of the enzymes.
Hence, the conditions for effective amplification were determined.
Although we did not test this complete module in B. subtilis, there are results available for the parts of this system. 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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