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 of restriction enzime for each circuit.
5. Based on the best combinantion design self-disruption plasmid.

Design

Restriction ensime candidates

Fse I

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.

I-SceI

Promoter candidates:

pLac

pBad

pRha

L-rhamnose-inducible promoter is capable of high-level recombinant protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose by the addition of D-glucose.

L-rhamnose is taken up by the RhaT transport system, converted to L-rhamnulose by an isomerase RhaA and then phosphorylated by a kinase RhaB. Subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase RhaD into dihydroxyacetone phosphate, which is metabolized in glycolysis, and L-lactaldehyde. The latter can be oxidized into lactate under aerobic conditions and be reduced into L-1,2-propanediol under unaerobic conditions.

The genes rhaBAD are organized in one operon which is controlled by the rhaPBAD promoter. This promoter is regulated by two activators, RhaS and RhaR, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhaBAD. If L-rhamnose is available, RhaR binds to the rhaPRS promoter and activates the production of RhaR and RhaS. RhaS together with L-rhamnose in turn binds to the rhaPBAD and the rhaPT promoter and activates the transcription of the structural genes. However, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. Therefore, only the rhaPBAD promoter has to be cloned upstream of the gene that is to be expressed. Full induction of rhaBAD transcription also requires binding of the CRP-cAMP complex, which is a key regulator of catabolite repression.

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 degradation efficiency of I-SceI (Clonned parts)

Experimental setup

To mesure digestion efficiency of I-SceI, we did a trasformation of two plasmids with different antibiotic resistance into NEB Turbo E.Coli strain:

• First plasmid:
• Second plasmid:

As result we expected to select colonies with both plasmid and

Transformation results:

Selection: Chloramphenicol plate First plasmid: pBad & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]	Selection: Ampicillin plate First plasmid: pBad & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]	Selection: Chloramphenicol & ampicillin plate First plasmid: pBad & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]
Chloramphenicol plate	Ampicillin plate	Chloramphenicol & ampicillin plate
Chloramphenicol plate	Ampicillin plate	Chloramphenicol & ampicillin plate

Control for plasmid compatibility:

Chloramphenicol plate	Ampicillin plate	Chloramphenicol & ampicillin plate
Chloramphenicol plate	Ampicillin plate	Chloramphenicol & ampicillin plate

Recovery in glucose:

Chloramphenicol plate	Ampicillin plate	Chloramphenicol & ampicillin plate
Chloramphenicol plate	Ampicillin plate	Chloramphenicol & ampicillin plate
Chloramphenicol plate	Ampicillin plate	Chloramphenicol & ampicillin plate

Results

Present your results

Mesuring of I-SceI efficiency (TUDelft parts)

Experimental setup

Describe the experiment

Selection: Chloramphenicol plate First plasmid: pBad & RBS & I-SceI ([] from TUDelft) [Cm] Second plasmid: I-SceI restriction site [Amp]	Selection: Ampicillin plate First plasmid: pBad & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]
Chloramphenicol plate	Ampicillin plate

Results

Present your results

Characterisation of pRha

Experimental setup

Describe the experiment

Results

Present your results

Summary

Related Parts and Links

• [http://partsregistry.org/Part:BBa_K175041 BBa_K175041] p(LacI) controlled I-SceI homing endonuclease generator, designed by Tim Weenink, group: iGEM09 TUDelft (2009-10-11)
• [http://partsregistry.org/Part:BBa_K175027 BBa_K175027] I-SceI restriction site, designed by Tim Weenink, group: iGEM09 TUDelft (2009-10-11)
• [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] Promoter (lacI regulated, lambda pL hybrid), designed by Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton, group: Registry (2003-01-31)

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)