Difference between revisions of "Team:UBonn HBRS/Description/Cloning"

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=== Esterase-Laccase group ===
 
=== Esterase-Laccase group ===
 
Enzymes which hydrolyse esters or oxidise phenols and aromatic compounds found in commercial inks. Their ability to attack ink particles is not well studied.
 
Enzymes which hydrolyse esters or oxidise phenols and aromatic compounds found in commercial inks. Their ability to attack ink particles is not well studied.
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{{UBonn_HBRS/table-start|class=with-caption}}
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<html>
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<thead>
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<tr>
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<td>Gene name</td>
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<td>Enzyme coded</td>
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<td>Part number</td>
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<td>Note</td>
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</tr>
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</thead>
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<tbody>
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<tr>
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<td>EstC2</td>
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<td>Esterase</td>
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<td>BBa_K2029007</td>
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<td><a href="https://www.ncbi.nlm.nih.gov/pubmed/21619698" target="_blank">https://www.ncbi.nlm.nih.gov/pubmed/21619698</a></td>
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</tr>
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<tr>
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<td>LipA</td>
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<td>Lipase</td>
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<td>BBa_K2029008</td>
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<td><a href="http://onlinelibrary.wiley.com/doi/10.1002/bit.10201/abstract" target="_blank">http://onlinelibrary.wiley.com/doi/10.1002/bit.10201/abstract</a></td>
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</tr>
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<tr>
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<td>Bpul</td>
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<td>Laccase</td>
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<td>BBa_K2029009</td>
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<td><a href="https://www.ncbi.nlm.nih.gov/nucleotide/388482909" target="_blank">https://www.ncbi.nlm.nih.gov/nucleotide/388482909</a></td>
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</tr>
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</tbody>
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</html>
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{{UBonn_HBRS/table-end}}
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<html><div class="caption"></html>
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'''Table 2. Genes and Enzymes of Esterase-Laccase group.'''
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<html></div></html>
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== General Strategy ==
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The primary target chassis organism is ''B. subtilis'', for which a secretory tag system was designed. At first ''E. coli'' was only intended to be used as the chassis for cloning, but as the project went on, E.coli was decided on as the backup system in case gene expression in B.subtilis fails.
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=== In B. subtilis ===
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As they express secretory proteins, GOIs require a signaling system in order to function in B.subtilis.
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{{UBonn_HBRS/image|url=//2016.igem.org/wiki/images/3/3c/T--UBonn_HBRS--Cloning_general_strategy_b_subtilis.png|alt=Strategy B. subtilis|class=100 borderless|caption-class=with-caption|caption=<strong>Figure 1. Cloning strategy in B. subtilis</strong>}}
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For this reason, two secretory tags are chosen to precede the genes: P34-sacB (Part: BBa_K2029001) and P34-nprb (BBa_K2029002). Both are constitutive in nature, which means they cannot be deactivated.
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 +
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=== In E. coli ===
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E.coli is intended to be the “Plan B” host organism, in case protein expression in B.subtilis cannot be achieved.
 +
 +
An inductive promoter – “pLac” – is coupled with a sequence coding for ribosome binding site (RBS.) This whole sequence, so called pLac-RBS, was synthesised at Integrated DNA Technologies (IDT), thanks to their sponsorship.
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 +
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{{UBonn_HBRS/image|url=|alt=Strategy E. coli|class=100 borderless|caption-class=with-caption|caption=<strong>Figure 1. Cloning strategy in E. coli</strong>}}
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== Composite “shuttle” plasmid backbone ==
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As the GOIs are intended to be put into both chassis – ''E. coli'' and ''B. subtilis'', a so-called “shuttle” backbone was developed to ensure compatibility with both species. This plasmid backbone - now given the nomenclature pSBbs1AK1 in the iGEM part registry – contains two different origins of replication, as well as confers resistance to two different antibiotics, one for each organism: against Kanamycin for ''B. subtilis'' and against Ampicillin for ''E. coli''.
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{{UBonn_HBRS/footer}}
 
{{UBonn_HBRS/footer}}

Revision as of 00:37, 20 October 2016

 

Cloning

General Abstract

The cloning aspect of this project is responsible for programming bacterial cells to secrete enzymes which can decouple ink particles from paper fibers.

To this end, genes coding for enzymes were synthesised and ligated into a plasmid backbone, which is designed for use in both Escherichia coli and Bacillus subtilis.

As expression of enzymes is intended for both Bacillus subtilis and E.coli, there are two separate tracks for cloning. For B. subtilis, the genes were assembled into pSBbs1AK1 along with secretory tags, and for E.coli, an inductive promoter (pLac) is placed upstream of the genes.


Genes of Interest

Within the scope of this project, genes of interest (GOIs) are those which catalyse either cellulolysis of paper fiber, or hydrolysis of ink particles.

Synthesised at GenScript, each of these genes includes a sequence coding for His-tag, for ease of protein purification.


Cellulase group

Enzymes which break down cellulose molecules into monosaccharides, therefore partially disintegrate paper fibers and set ink particles free into the supernatant.


Gene name Enzyme coded Part number Note
XynA Xylanase BBa_K2029002 http://www.microbialcellfactories.com/content/11/1/66
CelA Cellulase BBa_K2029004 https://www.ncbi.nlm.nih.gov/pubmed/22101447
EngB Endoglucanase BBa_K2029005 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1785254/
BsCel5c Cellulase BBa_K2029006 http://www.ncbi.nlm.nih.gov/pubmed/21549854
Table 1. Genes and Enzymes of Cellulase group.


Esterase-Laccase group

Enzymes which hydrolyse esters or oxidise phenols and aromatic compounds found in commercial inks. Their ability to attack ink particles is not well studied.


Gene name Enzyme coded Part number Note
EstC2 Esterase BBa_K2029007 https://www.ncbi.nlm.nih.gov/pubmed/21619698
LipA Lipase BBa_K2029008 http://onlinelibrary.wiley.com/doi/10.1002/bit.10201/abstract
Bpul Laccase BBa_K2029009 https://www.ncbi.nlm.nih.gov/nucleotide/388482909
Table 2. Genes and Enzymes of Esterase-Laccase group.


General Strategy

The primary target chassis organism is B. subtilis, for which a secretory tag system was designed. At first E. coli was only intended to be used as the chassis for cloning, but as the project went on, E.coli was decided on as the backup system in case gene expression in B.subtilis fails.


In B. subtilis

As they express secretory proteins, GOIs require a signaling system in order to function in B.subtilis.


Strategy B. subtilis
Figure 1. Cloning strategy in B. subtilis


For this reason, two secretory tags are chosen to precede the genes: P34-sacB (Part: BBa_K2029001) and P34-nprb (BBa_K2029002). Both are constitutive in nature, which means they cannot be deactivated.


In E. coli

E.coli is intended to be the “Plan B” host organism, in case protein expression in B.subtilis cannot be achieved.

An inductive promoter – “pLac” – is coupled with a sequence coding for ribosome binding site (RBS.) This whole sequence, so called pLac-RBS, was synthesised at Integrated DNA Technologies (IDT), thanks to their sponsorship.


Strategy E. coli
Figure 1. Cloning strategy in E. coli


Composite “shuttle” plasmid backbone

As the GOIs are intended to be put into both chassis – E. coli and B. subtilis, a so-called “shuttle” backbone was developed to ensure compatibility with both species. This plasmid backbone - now given the nomenclature pSBbs1AK1 in the iGEM part registry – contains two different origins of replication, as well as confers resistance to two different antibiotics, one for each organism: against Kanamycin for B. subtilis and against Ampicillin for E. coli.


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