Team:Paris Bettencourt/Restriction Enzyme

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Overview

Our group was responsible for designing self-plasmid digestion system. This synthetic system allows to digest plasmids into linear parts of DNA which afterwards could be degraded by Colicin.

Objectives

1. To find appropriate restriction enzymes which have to match the next properties:
   • In the E.Coli genome there is no restriction sites of a choosen restriction enzyme;
   • It has to have high specifity;
   • It have to works in wide range of different conditions (pH, T°C, etc)
2. Choose very strong promoter to regulate restriction enzyme expression;
3. To clone circuits with different combinations of choosen restriction enzymes and promoters.
4. Mesure degradation efficiency for each circuit.
5. Based on the best combinantion design self-disruption plasmid.

Design

On a role of restriction ensime we found two candidates:

1. Fse I - restriction endonucleases which recognize 8bp long DNA sequence: GGCCGG▽CC (CC△GGCCGG). The most important to methion that it has the lowest number of restriction sites in E.Coli genome: only 4 copies. We decided to use MAGE to remove it from chromosome (see for more detiles), but after MAGE did not have the expected yield, we decided to stop it and works only wuth the next candidate.
2. I-SceI

Promoter candidates:

1. pLac
2. pBad
3. pRha

As result we decided to clone such constructs in low-copy vector pSB3C5:

1. pLac & RBS & I-SceI
2. pBad & RBS & I-SceI
3. pRha & RBS & I-SceI

Also, as control vectors we cloned in the same back bone vector:

1. pLac & RBS & RFP
2. pBad & RBS & RFP
3. pRha & RBS & RFP

In our experiments we also used two biobricks which were send to us by TUDelf iGEM team:

1. [http://partsregistry.org/Part:BBa_K175027 BBa_K175027]
2. [http://partsregistry.org/Part:BBa_K175041 BBa_K175041]

Experiments and results

Mesuring of I-SceI efficiency (Clonned parts)

Experimental setup

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Results

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Mesuring of I-SceI efficiency (TUDelft parts)

Experimental setup

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Results

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Characterisation of pRha

Experimental setup

Describe the experiment

Results

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Summary

References

1. Janise Meyertons Nelson et al., «Fsel, a new type II restriction endonuclease that recognizes the octanucleotide sequence 5′ GGCCGGCC 3′»
2. Wernette C. M., «Structure and activity of the mitochondrial intron-encoded endonuclease, I-SceIV», Biochem Biophys Res Commun. 1998 Jul 9; 248(1):127-33.
3. Yisheng Kang et al., «Systematic Mutagenesis of E.coli K-12 MG1655 ORFs»
4. Jeanine M. Pennington, «On Spontaneous DNA Damage in Single Living Cells», Ph.D. thesis, Baylor College of Medicine, Houston (2006):
5. Susan M. Rosenberg, «A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation»
6. Colleaux et al., «Universal Code Equivalent of a Yeast Mitochondrial lntron Reading Frame Is Expressed into E. coli as a Specific Double Strand Endonuclease», (1986)