Difference between revisions of "Part:BBa K1444018"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K1444018 short</partinfo> | <partinfo>BBa_K1444018 short</partinfo> | ||
| − | Bacillus subtilis is a gram-positive bacterial species widely used in molecular biology labs around the world. It is capable of natural transformation under conditions of nutrient deprivation (energy starvation), and unlike many gram-negative species B. subtilis does not appear to require a specific uptake sequence. However, transformation through starvation may not be the most ideal process to implement in the lab as the protocols are very time-consuming, very sensitive to precise timings, and can be unreliable. Fortunately, all of the DNA uptake genes are under the control of a single transcription factor, so we can bypass the need for energy starvation by using cells derived from a specific strain of B. subtilis (SCK6) with the pAX01-comK plasmid constructed by | + | Bacillus subtilis is a gram-positive bacterial species widely used in molecular biology labs around the world. It is capable of natural transformation under conditions of nutrient deprivation (energy starvation), and unlike many gram-negative species B. subtilis does not appear to require a specific uptake sequence. However, transformation through starvation may not be the most ideal process to implement in the lab as the protocols are very time-consuming, very sensitive to precise timings, and can be unreliable. Fortunately, all of the DNA uptake genes are under the control of a single transcription factor, so we can bypass the need for energy starvation by using cells derived from a specific strain of B. subtilis (SCK6) with the pAX01-comK plasmid constructed by Zhang (2010) <cite>Zhang</cite>. |
This part contains a xylose-inducible promoter (pxylA), a ribosome binding site, and the comK coding sequence. This part is meant to be inserted into the genome of B. subtilis using an appropriate integration vector (unfortunately likely requiring the traditional transformation procedure). Once complete, you have a strain that can be transformed quickly and easily. | This part contains a xylose-inducible promoter (pxylA), a ribosome binding site, and the comK coding sequence. This part is meant to be inserted into the genome of B. subtilis using an appropriate integration vector (unfortunately likely requiring the traditional transformation procedure). Once complete, you have a strain that can be transformed quickly and easily. | ||
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It must be noted that this sequence still contains 1 SpeI and 2 EcoRI sites. As such, cloning with this part would necessitate using either XbaI and PstI, or NotI (it is a self-contained generator, so the directionality in the genome is not important). | It must be noted that this sequence still contains 1 SpeI and 2 EcoRI sites. As such, cloning with this part would necessitate using either XbaI and PstI, or NotI (it is a self-contained generator, so the directionality in the genome is not important). | ||
| − | + | ====References==== | |
| + | <biblio> | ||
| + | # Zhang Zhang, X-Z., & Zhang Y-H. (2010). Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1):98-105 | ||
| + | </biblio> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Revision as of 23:59, 15 October 2014
Xylose -> comK
Bacillus subtilis is a gram-positive bacterial species widely used in molecular biology labs around the world. It is capable of natural transformation under conditions of nutrient deprivation (energy starvation), and unlike many gram-negative species B. subtilis does not appear to require a specific uptake sequence. However, transformation through starvation may not be the most ideal process to implement in the lab as the protocols are very time-consuming, very sensitive to precise timings, and can be unreliable. Fortunately, all of the DNA uptake genes are under the control of a single transcription factor, so we can bypass the need for energy starvation by using cells derived from a specific strain of B. subtilis (SCK6) with the pAX01-comK plasmid constructed by Zhang (2010) Zhang.
This part contains a xylose-inducible promoter (pxylA), a ribosome binding site, and the comK coding sequence. This part is meant to be inserted into the genome of B. subtilis using an appropriate integration vector (unfortunately likely requiring the traditional transformation procedure). Once complete, you have a strain that can be transformed quickly and easily.
It must be noted that this sequence still contains 1 SpeI and 2 EcoRI sites. As such, cloning with this part would necessitate using either XbaI and PstI, or NotI (it is a self-contained generator, so the directionality in the genome is not important).
References
<biblio>
- Zhang Zhang, X-Z., & Zhang Y-H. (2010). Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1):98-105
</biblio>
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 557
Illegal EcoRI site found at 572
Illegal SpeI site found at 170 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 557
Illegal EcoRI site found at 572
Illegal SpeI site found at 170 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 557
Illegal EcoRI site found at 572
Illegal BamHI site found at 203 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 557
Illegal EcoRI site found at 572
Illegal SpeI site found at 170 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 557
Illegal EcoRI site found at 572
Illegal SpeI site found at 170 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 743