Difference between revisions of "Team:TU Darmstadt/Lab"

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<div class="abstract">
 
<div class="abstract">
 
<p><b>ABSTRACT</b><br/>
 
<p><b>ABSTRACT</b><br/>
<b>[...]</b></p>
+
<p><b>Abstract</b></p>
 +
 
 +
 
 +
<p>Artificial plasmids, which we transform into cells during the year on iGEM all the time,
 +
are a significant burden to the host. The design of our pathways, for example the combination of a promoter and RBS,
 +
result in different amounts of product. The measurement of the metabolic burden is the key for a quantitative optimization
 +
in metabolic engineering. Hereby, the measurement of the hosts' optical density, which should give you a feedback on the
 +
growth rate, shows you a very inaccurate value of the metabolic burden and even that just after a long time. F.&nbsp;Ceronie, R.&nbsp;Algar,
 +
GB.&nbsp;Stan, T.&nbsp;Ellis thought about the need of a highly quantitative accurate measurement and found a solution in the measurement of
 +
  a fluorophore, which the host expresses constitutively. They demonstrate, that the measurement of  GFP has great advantages
 +
over the measurement of OD, because it is much faster and more precise. Using this method, it is now possible to measure the
 +
impact of transformed plasmid live and with high accuracy. This new approach is of economical interest, because it enables
 +
scientist to test a lot of different pathways at once in a short time, just by using a fluoreader.</br>
 +
Our main project aims
 +
on developing a safety plasmid. To measure the metabolic burden caused by the safety plasmid, but also every plasmid that we
 +
design now and in the future, we want to build a meausurement strain based on the model of  F.&nbsp;Ceronie et al. To achieve the
 +
most sensitive results, we used the &lambda;&#8209;Integrase Site&#8209;specific Recombination Pathway, described by A.&nbsp;Landy
 +
in 2015, to integrate exactly one copy of GFP into <i>E.&nbsp;coli</i> K12 JM109. Therfore we designed two plasmids, based on
 +
BBa_I11020 and BBa_I11023. We measured our strain using single cell measurement as well as measurements with a fluoreader.</p>
 +
<p><b>Metabolic burden</b></p>
 +
<p> .......</p>
 +
 
 +
<p><b>Genomic integration</b></p>
 +
 
 +
<p> The <i>&lambda;</i>&#8209;integrase, originally derived from the <i>&lambda;</i>&#8209;Phage, catalyzes in combination with several assisting proteins the excessive and integrative recombination of the phage's genome with the chromosomal genome of a host.  For this, two <i>attachment sites</i> are needed: one located on the bacterial genome (<i>attB</i>) and the other located on the <i>&lambda;</i>&#8209;genome, which also contains several binding sites for regulatory proteins. The <i>attachment sites</i> contain homologous recognition sequences, called BOB'&nbsp;Region (<i>attB</i>) and COC'&nbsp;Region (<i>attP</i>). These can be connected by the <i>&lambda;</i>&#8209;integrase and the bacterial <i>integration host  factor</i> (IHF) via <i>Holliday junction</i> forming an intasome, a DNA&#8209;protein&#8209;complex, producing hybrid <i>attachment sites attL</i> and <i>attR</i>.
 +
<br>For the integration of a gene of interest (GOI) into the chromosomal genome of <i>E.&nbsp;coli</i> there are two plasmids needed. One, called integration plasmid, contains the constitutively expressed GOI GFP with a LVA degradation tag, which, as previously mentioned, is also the reporter that is necessary for the measurement of the metabolic burden and should be integrated into the <i>E.&nbsp;coli</i> genome. It also contains the <i>attP&nbsp;site</i> that enables the integration. There are two bidirectional terminators located on each side of the <i>attP</i> to protect the GFP&nbsp;Operon from the  transcription of the other neighbouring genes. The antibiotic resistance will also be integrated into the genome if the genomical integration succeeds, so we decided to use a Kanamycin resistance, as it is less commonly used in iGEM than Ampicillin or Chloramphenicol. Therefore, we chose the <i>backbone</i> pSB3K3, which also possesses a <i>low copy ori</i> and eases the later performed plasmid curing. The second plasmid is a helper plasmid and is necessary for transposing the GFP into the chromosomal genome as it contains the protein <i>&lambda;</i>&#8209;integrase with a <i>ribosomal binding site</i> (RBS).<br>To verify whether the recombination was successful one can perform a PCR with primers binding to the <i>attB site</i> of the <i>E.&nbsp;coli</i> and the VR Primer, which binds on every BioBrick compliant plasmid. As the one primer binds on the genome and the other on a plasmid, there can only be a PCR amplicon if the integration has succeeded.</p>
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 +
</p>
 
<button class="read_more" id="lab4b"><p>interested? read more</p></button>
 
<button class="read_more" id="lab4b"><p>interested? read more</p></button>
 
</div>
 
</div>

Revision as of 18:54, 2 October 2016

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iGEM TU Darmstadt 2016

IN THE LAB...

THE PROJECT
[...]

ORTHOGONAL PAIR

ABSTRACT
[...]

REPORTER

ABSTRACT
[...]

KILL(switch)

ABSTRACT
[...]

GENOMIC INTEGRATION

ABSTRACT

Abstract

Artificial plasmids, which we transform into cells during the year on iGEM all the time, are a significant burden to the host. The design of our pathways, for example the combination of a promoter and RBS, result in different amounts of product. The measurement of the metabolic burden is the key for a quantitative optimization in metabolic engineering. Hereby, the measurement of the hosts' optical density, which should give you a feedback on the growth rate, shows you a very inaccurate value of the metabolic burden and even that just after a long time. F. Ceronie, R. Algar, GB. Stan, T. Ellis thought about the need of a highly quantitative accurate measurement and found a solution in the measurement of a fluorophore, which the host expresses constitutively. They demonstrate, that the measurement of GFP has great advantages over the measurement of OD, because it is much faster and more precise. Using this method, it is now possible to measure the impact of transformed plasmid live and with high accuracy. This new approach is of economical interest, because it enables scientist to test a lot of different pathways at once in a short time, just by using a fluoreader.
Our main project aims on developing a safety plasmid. To measure the metabolic burden caused by the safety plasmid, but also every plasmid that we design now and in the future, we want to build a meausurement strain based on the model of F. Ceronie et al. To achieve the most sensitive results, we used the λ‑Integrase Site‑specific Recombination Pathway, described by A. Landy in 2015, to integrate exactly one copy of GFP into E. coli K12 JM109. Therfore we designed two plasmids, based on BBa_I11020 and BBa_I11023. We measured our strain using single cell measurement as well as measurements with a fluoreader.

Metabolic burden

.......

Genomic integration

The λ‑integrase, originally derived from the λ‑Phage, catalyzes in combination with several assisting proteins the excessive and integrative recombination of the phage's genome with the chromosomal genome of a host. For this, two attachment sites are needed: one located on the bacterial genome (attB) and the other located on the λ‑genome, which also contains several binding sites for regulatory proteins. The attachment sites contain homologous recognition sequences, called BOB' Region (attB) and COC' Region (attP). These can be connected by the λ‑integrase and the bacterial integration host factor (IHF) via Holliday junction forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites attL and attR.
For the integration of a gene of interest (GOI) into the chromosomal genome of E. coli there are two plasmids needed. One, called integration plasmid, contains the constitutively expressed GOI GFP with a LVA degradation tag, which, as previously mentioned, is also the reporter that is necessary for the measurement of the metabolic burden and should be integrated into the E. coli genome. It also contains the attP site that enables the integration. There are two bidirectional terminators located on each side of the attP to protect the GFP Operon from the transcription of the other neighbouring genes. The antibiotic resistance will also be integrated into the genome if the genomical integration succeeds, so we decided to use a Kanamycin resistance, as it is less commonly used in iGEM than Ampicillin or Chloramphenicol. Therefore, we chose the backbone pSB3K3, which also possesses a low copy ori and eases the later performed plasmid curing. The second plasmid is a helper plasmid and is necessary for transposing the GFP into the chromosomal genome as it contains the protein λ‑integrase with a ribosomal binding site (RBS).
To verify whether the recombination was successful one can perform a PCR with primers binding to the attB site of the E. coli and the VR Primer, which binds on every BioBrick compliant plasmid. As the one primer binds on the genome and the other on a plasmid, there can only be a PCR amplicon if the integration has succeeded.

CHEMICAL SYNTHESIS

ABSTRACT
[...]