Difference between revisions of "Part:BBa K1444018"
 
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<partinfo>BBa_K1444018 short</partinfo>
 
<partinfo>BBa_K1444018 short</partinfo>
  
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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) <cite>Zhang</cite>.
+
Xylose-induced activation of the ''B. subtilis'' natural transformation system.
  
−
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.  
+
 
 +
<!-- -->
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K1444018 SequenceAndFeatures</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 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).
 
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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====References====
 
−
<biblio>
 
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# 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>
 
  
 
===Usage and Biology===
 
===Usage and Biology===
 
Though the DNA was not submitted to the iGEM registry, we have characterized the strain that we amplified the part from.
 
Though the DNA was not submitted to the iGEM registry, we have characterized the strain that we amplified the part from.
−
====Protocol====
+
====Transformation protocol====
 
The protocol is described in detail [http://2014.igem.org/Team:Calgary/Notebook/ProtocolManual/Bsubtilis here], but in brief:<br>
 
The protocol is described in detail [http://2014.igem.org/Team:Calgary/Notebook/ProtocolManual/Bsubtilis here], but in brief:<br>
−
*Grow ''B. subtilis'' overnight at 37 C with shaking
+
*Grow ''B. subtilis'' overnight at 37&nbsp;°C with shaking
−
*Subculture to an OD of 1.0
+
*Dilute the culture to an OD<sub>600</sub> of 1.0
 
*Add xylose dissolved in LB to a final concentration (w/v) of 2%
 
*Add xylose dissolved in LB to a final concentration (w/v) of 2%
 
*Grow for 2 hours with shaking
 
*Grow for 2 hours with shaking
Line 25: Line 29:
 
*Plate overnight with appropriate selection
 
*Plate overnight with appropriate selection
  
 +
====Diluting a ''B. subtilis'' culture====
 +
[[File:Calgary_absorb_standard_curve.png|500px|thumb|right|'''Figure 1:''' The decrease in optical density from addition of fresh LB media to an overnight culture of ''B. subtilis''.]]
  
 +
<p align="left">
 +
The second step in the protocol above calls for the overnight culture of ''B. subtilis''. This can be rather tedious, so we generated a standard curve of how the OD<sub>600</sub> decreased with the addition of LB media. 500 &micro;L of an overnight culture was diluted, and the decrease in OD<sub>600</sub> was noted. This allows a user to measure the cell density of their overnight culture, and then consult the graph to determine how much fresh LB must be added to drop the absorbance to 1.0.
 +
</p>
 +
<br style="clear: both" />
 +
====Effects of temperature and xylose====
 +
[[File:Calgary_xylose_conc_temp_graph.png|400px|thumb|right|'''Figure 2:''' The effects of temperature and xylose concentration on the transformation efficiency of ''B. subtilis'' on LB plates.]]
 +
[[File:Calgary_xylose_od_graph.png|400px|thumb|right|'''Figure 3:''' The effects of xylose concentration on the transformation efficiency of ''B. subtilis'' in small volume liquid culture.]]
  
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<!-- -->
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<p align="left">
−
<span class='h3bb'>Sequence and Features</span>
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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.
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<partinfo>BBa_K1444018 SequenceAndFeatures</partinfo>
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</p>
 +
<p align="left">
 +
We also repeated the test in small volume liquid culture, by transforming ''B. subtilis'' to yield a final volume of 100 &micro;L after addition of xylose, and measuring the cell density via OD<sub>600</sub> after approximately 20 hours.
 +
</p>
 +
<br style="clear: both" />
 +
===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
 +
 
 +
 
 +
===Improvement of Part - UofC_Calgary 2016===
 +
The UofC_Calgary 2016 iGEM team improved this part by removing illegal internal restriction sites: one SpeI site located at base pairs 170-175 and two EcoRI sites located at base pairs 557-562 & 572-577. The SpeI site (ACTAGT) was deleted entirely, as the site was contained within a spacer region between the xylose-inducible promoter and RBS. To remove EcoRI sites, the DNA bases of the new comK construct were changed, and codons were optimized for <i>B. subtilis</i>:<br><br>
 +
 
 +
553 A>G<br>
 +
556 C>T<br>
 +
568 A>G<br>
 +
571 C>T<br><br>
 +
 
 +
This improved part can be found here: https://parts.igem.org/Part:BBa_K2008006<br>
 +
Note: sequence for this part was submitted to the registry in 2016.<br><br>
 +
 
 +
Additionally, the UofC_Calgary 2016 iGEM team added flanking regions of homology on either end of the <i>comK</i> construct. These regions of homology were derived from the <i>B. subtilis amyE</i> locus to allow for chromosomal integration.
  
 +
This improved part can be found here: https://parts.igem.org/Part:BBa_K2008007<br>
 +
Note: sequence for this part was submitted to the registry in 2016.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 20:00, 18 October 2016

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
  • Dilute the culture to an OD600 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

Diluting a B. subtilis culture

Figure 1: The decrease in optical density from addition of fresh LB media to an overnight culture of B. subtilis.

The second step in the protocol above calls for the overnight culture of B. subtilis. This can be rather tedious, so we generated a standard curve of how the OD600 decreased with the addition of LB media. 500 µL of an overnight culture was diluted, and the decrease in OD600 was noted. This allows a user to measure the cell density of their overnight culture, and then consult the graph to determine how much fresh LB must be added to drop the absorbance to 1.0.


Effects of temperature and xylose

Figure 2: The effects of temperature and xylose concentration on the transformation efficiency of B. subtilis on LB plates.
Figure 3: The effects of xylose concentration on the transformation efficiency of B. subtilis in small volume liquid culture.

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.

We also repeated the test in small volume liquid culture, by transforming B. subtilis to yield a final volume of 100 µL after addition of xylose, and measuring the cell density via OD600 after approximately 20 hours.


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


Improvement of Part - UofC_Calgary 2016

The UofC_Calgary 2016 iGEM team improved this part by removing illegal internal restriction sites: one SpeI site located at base pairs 170-175 and two EcoRI sites located at base pairs 557-562 & 572-577. The SpeI site (ACTAGT) was deleted entirely, as the site was contained within a spacer region between the xylose-inducible promoter and RBS. To remove EcoRI sites, the DNA bases of the new comK construct were changed, and codons were optimized for B. subtilis:

553 A>G
556 C>T
568 A>G
571 C>T

This improved part can be found here: https://parts.igem.org/Part:BBa_K2008006
Note: sequence for this part was submitted to the registry in 2016.

Additionally, the UofC_Calgary 2016 iGEM team added flanking regions of homology on either end of the comK construct. These regions of homology were derived from the B. subtilis amyE locus to allow for chromosomal integration.

This improved part can be found here: https://parts.igem.org/Part:BBa_K2008007
Note: sequence for this part was submitted to the registry in 2016.