Team:Imperial College London/Chassis

From 2010.igem.org

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{| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
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|-
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  ! Required components !! Features of ''B. subtilis'
 +
|-
 +
  |For our '''detection module''' we need:
 +
 +
* A peptide display system combined with
 +
 +
* A quorum sensing system activated by a linear AIP.
 +
 +
 +
For our chassis we need it to be '''Portable'''
 +
 +
||
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* Utilizes a quorum sensing system based on using small linear  peptides as autoinducers
 +
 +
* LytC peptide delivery and cell wall anchoring system
 +
 +
* Bacillus contains a single cell membrane which alleviates additional complexity transporting our re-engineered surface peptide between compartments
 +
 +
* Spore forming under stressful conditions
 +
 +
* High motility for faster detection of the parasite!
 +
 +
|}
 +
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'''Notes:'''
 +
Bacillus uniquely utilizes a quorum sensing system based on using small peptides as autoinducers. Many other bacteria including gram negative bacteria use a QSS based on circular or post-translationally modified peptide display systems; this introduced problems with our desire to re-engineer the small peptide fragment. Bacillus however presents linear peptides on its cell wall. Small autoinducing peptides of a downstream signaling system could be released by the cleavage of these membrane-anchored cell surface proteins. This involves including a protease cleavage site between the membrane anchored domain and the autoinducing peptide. Proteases released from the Circaria parasite would target this site and hence we can detect them as our system becomes activated.
 +
Protease detector peptides (small linear quorum sensing peptide attached to protease recognition sequence) may also get trapped between the cell wall and cell membrane. To avoid this we looked into mechanisms of transport and attachment of these peptides to the outside of cell wall. An alternative was secrete detector peptides into the medium as B.subtilis is very good at secreting proteins. The lytC cell wall delivery and anchoring system was chosen to satisfy our system of peptide presentation. Specifically, our protease cleavage site linker peptide plus autoinducing peptide were attached to the cell wall binding domain of an endogenous (to Bacillus) LytC surface protein.
 +
 +
NB. An additional option considered was to attach the small protease detector peptides on to pili (fimbriae) that are normally presented on the bacterial cell. This has been done routinely on E.coli fimbriae at non-conserved regions and outer membrane proteins to present various antigens [[1]]. There is also a good review of a variety of export patways that have been used [[2]] in gram negative bacteria. There is a lot work done on gram positive bacteria, although there is a detailed and very accessible review [[3]] of current understanding of G+ve cell surface.
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{| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
 +
 +
|-
 +
  ! Issues:!! Solution:
 +
|-
 +
  |Extracellular proteases naturally produced and secreted by gram positive bacteria.
 +
''These could interfere with the system and could result in false positives or degrade the protease detection peptide.''
 +
|| Protease deficient strains are available
 +
 +
|}
 +
 +
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'''Safety'''
 +
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{| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
 +
 +
|-
 +
  ! Required components !! Features of ''B. subtilis'
 +
|-
 +
  |Our system is to be utilized in the field by non experts and to used in the environment,
 +
So we need to ensure the bacterium is '''SAFE'''
 +
|| Non pathogenic
 +
 +
|}
 +
 +
'''Notes:'''
 +
Bacillus is a naturally occurring soil bacterium that presents no pathogenicity to humans. We aim to introduce our new DNA into existing essential genes. The disruption of these genes render the bacteria unlikely to exist outside of the lab or eventual product environment.
 +
 +
 +
 +
 +
'''Design Considerations'''
 +
{| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
 +
 +
|-
 +
  ! Required components !! Features of ''B. subtilis'
 +
|-
 +
  | Integration of DNA into the host genome||
 +
* Natural Competency and Integration
 +
* Very well characterized Gram +ve bacterium
 +
 +
|}
 +
 +
'''Notes:'''
 +
Luckily for us B.subtilis was the chassis of choice of the IC iGEM 2008 team. This means that there are now Bacillus friendly parts in the registry that we can use and also further characterize and contribute towards.
 +
 +
 +
{| class="wikitable" style="text-align: center; width: 50%; height: 170px;" border="1"
 +
 +
|-
 +
  ! Required components !! Features of ''B. subtilis'
 +
|-
 +
  |
 +
* Recognition of vectors as foreign DNA and subsequent rejection
 +
* Vector incompatibility with those used by ''E.coli''
 +
|| *Chromosome integration methods, ultimately ensures our introduced DNA is more stable and retained.
 +
 +
|}
 +
 +
[[Image:IC_2010_IGEMchassis.jpg|thumb|center|800px]]
[[Image:IC_2010_IGEMchassis.jpg|thumb|center|800px]]
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===<big>''B. subtilis'' ''dif'': sequence-specific recombinase recognition site</big>===
===<big>''B. subtilis'' ''dif'': sequence-specific recombinase recognition site</big>===
-
In cells with circular chromosomes, recombinatorial repair and homologous recombination can generate multimeric chromosomes <cite>1</cite>. ‘’Dif’’ sites are part of a system to ensure that multimeric chromosomes can be separated to monomers, which is required for proper sharing of genetic material between daughter cells.
+
In cells with circular chromosomes, recombinatorial repair and homologous recombination can generate multimeric chromosomes <cite>1</cite>. ''Dif'' sites are part of a system to ensure that multimeric chromosomes can be separated to monomers, which is required for proper sharing of genetic material between daughter cells.
-
In ‘’B. subtilis’’ the tyrosine family recombinases such as RipX and CodV mediate the separation at 28-bp sequence Bs’’dif’’ <cite>1</cite>. The site-specific recombinases are able to recognize two directly repeated ’’dif’’ sites and excise the fragment flanked by the two sites <cite>2</cite>.
+
In ''B. subtilis'' the tyrosine family recombinases such as RipX and CodV mediate the separation at 28-bp sequence Bs ''dif'' <cite>1</cite>. The site-specific recombinases are able to recognize two directly repeated ''dif'' sites and excise the fragment flanked by the two sites <cite>2</cite>.
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The site-specific recombinases, endogenous to ‘’B. subtilis’’ strains are able to recognise Bs’’dif’’ sites and recombine out the strand of DNA directly flanked by the two sites. Recombination leaves a single dif site.
The site-specific recombinases, endogenous to ‘’B. subtilis’’ strains are able to recognise Bs’’dif’’ sites and recombine out the strand of DNA directly flanked by the two sites. Recombination leaves a single dif site.
-
The construct was previously engineered to homologously recobine into the genome of ‘’B. subtilis’’. Integration sequences such as amyE <bbpart>BBa_K143001</bbpart>, <bbpart>BBa_K143002</bbpart> can be used to achieve this.  
+
The construct was previously engineered to homologously recobine into the genome of ''B. subtilis''. Integration sequences such as amyE <bbpart>BBa_K143001</bbpart>, <bbpart>BBa_K143002</bbpart> can be used to achieve this.  

Revision as of 17:13, 24 October 2010

Chassis

Our choice of chassis was B. subtilis.

Icbsub.jpg

Here we list the reasons why B. subtilis serves as a more appropriate chassis option with regards to our project design and main project specifications. To re-iterate the pertinent project specifications:

  • Safe
  • Portable
  • Cheap
  • Fast
  • Easy to use

We selected B. subtilis because it is a well characterized gram positive bacterium that is non pathogenic. It was convenient but also a coincidence that within our chosen chassis were features with which we could manipulate to use in our detection module.

Below is a brief table summary of how Bacillus meets our system requirements and also some of the issues we encountered throughout the implementation process and how we overcame them.

Bacillus | Breakdown


Required components Features of B. subtilis'
For our detection module we need:
  • A peptide display system combined with
  • A quorum sensing system activated by a linear AIP.


For our chassis we need it to be Portable 

* Utilizes a quorum sensing system based on using small linear  peptides as autoinducers
  • LytC peptide delivery and cell wall anchoring system
  • Bacillus contains a single cell membrane which alleviates additional complexity transporting our re-engineered surface peptide between compartments
  • Spore forming under stressful conditions
  • High motility for faster detection of the parasite!

Notes: Bacillus uniquely utilizes a quorum sensing system based on using small peptides as autoinducers. Many other bacteria including gram negative bacteria use a QSS based on circular or post-translationally modified peptide display systems; this introduced problems with our desire to re-engineer the small peptide fragment. Bacillus however presents linear peptides on its cell wall. Small autoinducing peptides of a downstream signaling system could be released by the cleavage of these membrane-anchored cell surface proteins. This involves including a protease cleavage site between the membrane anchored domain and the autoinducing peptide. Proteases released from the Circaria parasite would target this site and hence we can detect them as our system becomes activated. Protease detector peptides (small linear quorum sensing peptide attached to protease recognition sequence) may also get trapped between the cell wall and cell membrane. To avoid this we looked into mechanisms of transport and attachment of these peptides to the outside of cell wall. An alternative was secrete detector peptides into the medium as B.subtilis is very good at secreting proteins. The lytC cell wall delivery and anchoring system was chosen to satisfy our system of peptide presentation. Specifically, our protease cleavage site linker peptide plus autoinducing peptide were attached to the cell wall binding domain of an endogenous (to Bacillus) LytC surface protein.

NB. An additional option considered was to attach the small protease detector peptides on to pili (fimbriae) that are normally presented on the bacterial cell. This has been done routinely on E.coli fimbriae at non-conserved regions and outer membrane proteins to present various antigens 1. There is also a good review of a variety of export patways that have been used 2 in gram negative bacteria. There is a lot work done on gram positive bacteria, although there is a detailed and very accessible review 3 of current understanding of G+ve cell surface.



Issues: Solution:
Extracellular proteases naturally produced and secreted by gram positive bacteria.

These could interfere with the system and could result in false positives or degrade the protease detection peptide.

Protease deficient strains are available


Safety

Required components Features of B. subtilis'
Our system is to be utilized in the field by non experts and to used in the environment,

So we need to ensure the bacterium is SAFE

Non pathogenic

Notes: Bacillus is a naturally occurring soil bacterium that presents no pathogenicity to humans. We aim to introduce our new DNA into existing essential genes. The disruption of these genes render the bacteria unlikely to exist outside of the lab or eventual product environment.



Design Considerations

Required components Features of B. subtilis'
Integration of DNA into the host genome
  • Natural Competency and Integration
  • Very well characterized Gram +ve bacterium

Notes: Luckily for us B.subtilis was the chassis of choice of the IC iGEM 2008 team. This means that there are now Bacillus friendly parts in the registry that we can use and also further characterize and contribute towards.


Required components Features of B. subtilis'
  • Recognition of vectors as foreign DNA and subsequent rejection
  • Vector incompatibility with those used by E.coli
*Chromosome integration methods, ultimately ensures our introduced DNA is more stable and retained.


IC 2010 IGEMchassis.jpg


<partinfo>BBa_K316002 short</partinfo>

B. subtilis dif: sequence-specific recombinase recognition site

In cells with circular chromosomes, recombinatorial repair and homologous recombination can generate multimeric chromosomes 1. Dif sites are part of a system to ensure that multimeric chromosomes can be separated to monomers, which is required for proper sharing of genetic material between daughter cells. In B. subtilis the tyrosine family recombinases such as RipX and CodV mediate the separation at 28-bp sequence Bs dif 1. The site-specific recombinases are able to recognize two directly repeated dif sites and excise the fragment flanked by the two sites 2.


DifExcision.PNG

Figure 1. Removal of a specific gene from a genome integrated construct.

The site-specific recombinases, endogenous to ‘’B. subtilis’’ strains are able to recognise Bs’’dif’’ sites and recombine out the strand of DNA directly flanked by the two sites. Recombination leaves a single dif site.

The construct was previously engineered to homologously recobine into the genome of B. subtilis. Integration sequences such as amyE <bbpart>BBa_K143001</bbpart>, <bbpart>BBa_K143002</bbpart> can be used to achieve this.


Sequence and Features

<partinfo>BBa_K316002 SequenceAndFeatures</partinfo>



References

1) Sciochetti SA, Piggot PJ, and Blakely GW. Identification and characterization of the dif Site from Bacillus subtilis. J Bacteriol 2001 Feb; 183(3) 1058-68. doi:10.1128/JB.183.3.1058-1068.2001 pmid:11208805. PubMed HubMed

2) Bloor AE and Cranenburgh RM. An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol 2006 Apr; 72(4) 2520-5. doi:10.1128/AEM.72.4.2520-2525.2006 pmid:16597952.


E.coli was considered as a possible option. Despite the gram negative outer membrane, there are strains that have been made more permeable through knockout of Lipid A biosynthesis in lipopolysaccharides [4] There are also proteins that can disrupt membranes when they are inserted in them, thus making them more permeable. These chages in permeability are likely due to transient ruptures of outer membrane and so unlikely to make a very responsive or robust detecton organism [4].

References

Surplus information on B. Subtilis chassis [http://www.subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page Subti-Wiki] 

[http://mic.sgmjournals.org/cgi/content/full/146/12/3025 1]
[http://www.microbialcellfactories.com/content/5/1/22 2]
[http://www3.interscience.wiley.com/cgi-bin/fulltext/118603263/PDFSTART 3]
[http://aac.asm.org/cgi/content/full/43/6/1459?view=long&pmid=10348770 4]
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