Difference between revisions of "Team:NRP-UEA-Norwich/BioBricks"

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In order to assess whether our BioBricks would function in our chassis organism Shewanella oneidensis MR-1 we attempted to transform and express two BioBricks into S. oneidensis MR-1; J04450 containing a gene for the red fluorescent protein (RFP) and K584001 which contains a gene for the green fluorescent protein (GFP). These BioBricks were chosen as they contain reporter genes and successfully transformed colonies could be easily identified under UV light.
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In order to assess whether our BioBricks would function in our model organism <i>Shewanella oneidensis</i> MR-1 we attempted to transform and express two BioBricks into <i>S. oneidensis</i> MR-1; J04450 containing a gene for the red fluorescent protein (RFP) and K584001 which contains a gene for the green fluorescent protein (GFP). These BioBricks were chosen as they contain reporter genes and successfully transformed colonies could be easily identified under UV light.
 
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As S. oneidensis MR-1 are not amenable to heat shock transformation we took a tri-parental conjugation approach. We followed protocol 2:1. Parent Escherichia coli strains were transformed with J04450 and K584001 respectively using a heat shock approach according to protocol 0.2. For step 9 chloramphenicol was used instead of kanamycin as we were working with BioBricks and these contain chloramphenicol as a selective marker, not kanamycin. Carbenicillin was still used to select against E. coli parent and helper strains as S. oneidensis have natural resistance to this antibiotic.
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As <i>S. oneidensis</i> MR-1 are not able to undergo heat shock transformation we used a tri-parental conjugation approach (see protocol). Parent <i>Escherichia coli</i> strains contained J04450 and K584001 respectively using a heat shock approach according to protocol. For step 9 in the tri-parental conjugation protocol chloramphenicol was used instead of kanamycin as BioBricks contain chloramphenicol resistance as a selective marker instead of kanamycin.  
 
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After serial dilutions for all attempted conjugations no individual S. oneidensis MR-1 colonies survived, and therefore the cells failed to display chloramphenicol resistance. Escherichia coli parent and helper strains survived, so carbenicillin resistance was successfully transferred from S. oneidensis MR-1 to E. coli. This indicated conjugation must have been successful, however S. oneidensis MR-1 failed to display chloramphenicol resistance. As the promoters for the antibiotic resistance genes are constitutive the genes should have been transcribed if present. The origin of replication of these plasmids however, pMB1, have not been used in S. oneidensis MR-1 by past iGEM teams. The Harvard 2008 iGEM team did not list pMB1 as an effective origin of replication for plasmids in S. oneidensis MR-1. Additionally NTU Singapore in 2015 used the pHG101 plasmid which has a different origin of replication to allow for expression of their BioBrick part in S. oneidensis MR-1. Similarly as a result of our failure to express functional BioBricks in S. oneidensis MR-1 we decided to transfer our gene cluster to a modified pBAD vector using a golden gate cloning strategy. Our bio-bricks were designed to assemble the entire gene cluster for each separate hydrogenase into a single expression vector.
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After serial dilutions for all attempted conjugations no individual <i>S. oneidensis</i> MR-1 colonies survived, and therefore the cells failed to display chloramphenicol resistance. <i>E.coli</i> parent and helper strains survived, so carbenicillin resistance could have been transferred from <i>S. oneidensis</i> MR-1 to <i>E. coli</i>. Also possible however is that E. coli developed spontaneous carbenicillin resistance as they can easily gain mutations in the carbenicillin target protein, inferring resistance.  
 
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<p><i>S. oneidensis</i> MR-1 failed to display chloramphenicol resistance. As the promoters for the antibiotic resistance genes are constitutive the genes should have been transcribed if present. The origin of replication of these plasmids however, pMB1, has not been used in <i>S. oneidensis</i> MR-1 by past iGEM teams. The Harvard 2008 iGEM team did not list pMB1 as an effective origin of replication for plasmids in <i>S. oneidensis</i> MR-1. Additionally NTU Singapore in 2015 used the pHG101 plasmid which has a different origin of replication to allow for expression of their BioBrick part in <i>S. oneidensis</i> MR-1. Similarly as a result of our failure to express functional BioBricks in <i>S. oneidensis</i> MR-1 we decided to transfer our gene cluster to a modified pBAD vector for expression trials. With this information we designed our BioBricks to be assembled through a Golden Gate cloning strategy into any expression vector containing <i>Bsal</i> restriction sites. See the golden gate cloning page for more details. Our chosen pBAD vectors have been used in <i>S. oneidensis</i> before successfully (Edwards <i>et al.</i> 2015) making it a good choice for expression of our gene cluster.  
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In the future iGEM teams should investigate this to confirm the origin of replication is in fact the issue with BioBrick expression in S. oneidensis MR-1. If this is in fact the case future iGEM teams can then move on to apply synthetic biology to optimise the origin of replication of some BioBricks for the highest possible copy number in S. oneidensis MR-1. These can then serve as a backbone for the cloning of further genes to be expressed in S. oneidensis MR-1. This exercise will compound in value as this organism becomes more widely used for synthetic biology.  
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<p>In the future iGEM teams should investigate this to confirm the origin of replication is in fact the issue with BioBrick expression in <i>S. oneidensis</i> MR-1. If this is the case future iGEM teams can then move on to apply synthetic biology to optimise the origin of replication of some BioBricks for the highest possible copy number in <i>S. oneidensis</i> MR-1. These can then serve as a backbone for the cloning of further genes to be expressed in <i>S. oneidensis</i> MR-1 allowing more detailed investigations into the biotech applications of this organism.
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<div class="subTitle">References</div>
 
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<a href="https://2008.igem.org/Team:Harvard">https://2008.igem.org/Team:Harvard</a><br />
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‘Bactricity’ Harvard iGEM 2008<a href="https://2008.igem.org/Team:Harvard">https://2008.igem.org/Team:Harvard</a><br /><br/>
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<a href="https://2015.igem.org/Team:NTU-Singapore">https://2015.igem.org/Team:NTU-Singapore</a>
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NTU Singapore iGEM 2015 <a href="https://2015.igem.org/Team:NTU-Singapore">https://2015.igem.org/Team:NTU-Singapore</a> <br /><br/>
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Edwards <i>et al.</i> (2015) ‘Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer’, Scientific Report, [Online] DOI: 10.1038/srep11677
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Latest revision as of 18:28, 19 October 2016

NRP-UEA-NORWICH iGEM

BIOBRICKS

Aim

In order to assess whether our BioBricks would function in our model organism Shewanella oneidensis MR-1 we attempted to transform and express two BioBricks into S. oneidensis MR-1; J04450 containing a gene for the red fluorescent protein (RFP) and K584001 which contains a gene for the green fluorescent protein (GFP). These BioBricks were chosen as they contain reporter genes and successfully transformed colonies could be easily identified under UV light.

Methods

As S. oneidensis MR-1 are not able to undergo heat shock transformation we used a tri-parental conjugation approach (see protocol). Parent Escherichia coli strains contained J04450 and K584001 respectively using a heat shock approach according to protocol. For step 9 in the tri-parental conjugation protocol chloramphenicol was used instead of kanamycin as BioBricks contain chloramphenicol resistance as a selective marker instead of kanamycin.

Results

After serial dilutions for all attempted conjugations no individual S. oneidensis MR-1 colonies survived, and therefore the cells failed to display chloramphenicol resistance. E.coli parent and helper strains survived, so carbenicillin resistance could have been transferred from S. oneidensis MR-1 to E. coli. Also possible however is that E. coli developed spontaneous carbenicillin resistance as they can easily gain mutations in the carbenicillin target protein, inferring resistance.

S. oneidensis MR-1 failed to display chloramphenicol resistance. As the promoters for the antibiotic resistance genes are constitutive the genes should have been transcribed if present. The origin of replication of these plasmids however, pMB1, has not been used in S. oneidensis MR-1 by past iGEM teams. The Harvard 2008 iGEM team did not list pMB1 as an effective origin of replication for plasmids in S. oneidensis MR-1. Additionally NTU Singapore in 2015 used the pHG101 plasmid which has a different origin of replication to allow for expression of their BioBrick part in S. oneidensis MR-1. Similarly as a result of our failure to express functional BioBricks in S. oneidensis MR-1 we decided to transfer our gene cluster to a modified pBAD vector for expression trials. With this information we designed our BioBricks to be assembled through a Golden Gate cloning strategy into any expression vector containing Bsal restriction sites. See the golden gate cloning page for more details. Our chosen pBAD vectors have been used in S. oneidensis before successfully (Edwards et al. 2015) making it a good choice for expression of our gene cluster.

In the future iGEM teams should investigate this to confirm the origin of replication is in fact the issue with BioBrick expression in S. oneidensis MR-1. If this is the case future iGEM teams can then move on to apply synthetic biology to optimise the origin of replication of some BioBricks for the highest possible copy number in S. oneidensis MR-1. These can then serve as a backbone for the cloning of further genes to be expressed in S. oneidensis MR-1 allowing more detailed investigations into the biotech applications of this organism.

References

‘Bactricity’ Harvard iGEM 2008https://2008.igem.org/Team:Harvard

NTU Singapore iGEM 2015 https://2015.igem.org/Team:NTU-Singapore

Edwards et al. (2015) ‘Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer’, Scientific Report, [Online] DOI: 10.1038/srep11677

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