Difference between revisions of "Part:BBa K1444018"
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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).
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''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).
  
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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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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).
 
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).
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===Usage and Biology===
 
===Usage and Biology===
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====Effects of temperature and xylose====
 
====Effects of temperature and xylose====
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[[File:Calgary_xylose_conc_temp_graph.png|300px|thumb|right|'''Figure 1''': Test caption]]
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[[File:Calgary_xylose_conc_temp_graph.png|300px|thumb|right|'''Figure 1''': The effects of temperature and xylose concentration on the transformation efficiency of ''B. subtilis''.]]
  
 
<p align="left">
 
<p align="left">
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We have tested the original transformation protocol, and characterized several aspects of it.
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We have tested the original transformation protocol, and characterized several aspects of it. Here, we modified the concentration of xylose that the ''B. subtilis'' cells were exposed to, as well as the temperature that they were cultured in after addition of linearized plasmid DNA containing an antibiotic resistance cassette. After a 2-hour period, the cultures were spread onto selective media, and the colonies counted.
 
</p>
 
</p>
  
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====References====
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===References===
 
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
 
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
  

Revision as of 21:30, 16 October 2014

Xylose -> comK

Xylose-induced activation of the B. subtilis natural transformation system.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 557
    Illegal EcoRI site found at 572
    Illegal SpeI site found at 170
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 557
    Illegal EcoRI site found at 572
    Illegal SpeI site found at 170
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 557
    Illegal EcoRI site found at 572
    Illegal BamHI site found at 203
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 557
    Illegal EcoRI site found at 572
    Illegal SpeI site found at 170
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 557
    Illegal EcoRI site found at 572
    Illegal SpeI site found at 170
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 743


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).

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).


Usage and Biology

Though the DNA was not submitted to the iGEM registry, we have characterized the strain that we amplified the part from.

Transformation protocol

The protocol is described in detail [http://2014.igem.org/Team:Calgary/Notebook/ProtocolManual/Bsubtilis here], but in brief:

  • Grow B. subtilis overnight at 37 °C with shaking
  • Subculture to an OD of 1.0
  • Add xylose dissolved in LB to a final concentration (w/v) of 2%
  • Grow for 2 hours with shaking
    • Freeze aliquots if desired at this point
  • Add DNA (linearized plasmid or PCR product), grow for 2 hours with NO shaking
  • Plate overnight with appropriate selection

Effects of temperature and xylose

Figure 1: The effects of temperature and xylose concentration on the transformation efficiency of B. subtilis.

We have tested the original transformation protocol, and characterized several aspects of it. Here, we modified the concentration of xylose that the B. subtilis cells were exposed to, as well as the temperature that they were cultured in after addition of linearized plasmid DNA containing an antibiotic resistance cassette. After a 2-hour period, the cultures were spread onto selective media, and the colonies counted.



References

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