Part:BBa_K316010
Reverse strand coding XylE under LacI activation
This part contains 3' coding version of XylE BBa_J33204 under synthetic hyperspank promoter BBa_K316000. Constitutively expressed LacI such as BBa_K143033 is required for repression and IPTG for removal of expression.
In many biological settings there may be read-through caused by upstream elements, however this can often be unidirectional. In settings where readthrough from 5' direction is expected to be much stronger than 3' direction, it may is advantageous to use 3' coding sequence instead of traditional 5'. As transformation of B subtilis often requires genomic integration, read-through from upstream genomic regions can become an issue.
This part contains XylE coding on the 3' strand. For the purposes of characterisation, the part was made on the minus strand to reduce background transcription. Read-through from 5' direction may provide an additional benefit. Low quantities of anti-sence RNA may be produced as the result of 5' readthrough, which may in turn reduce basal expression sometimes seen in LacI systems.
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 3' coding XylE construct with associated Pehs BBa_K316000 promoter-RBS.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1076
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 437
Illegal NgoMIV site found at 609
Illegal AgeI site found at 86 - 1000COMPATIBLE WITH RFC[1000]
Please Note, the above parts are coding on the 3' strand in the opposite direction to the arrows shown, as per Figure I
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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