Team:Paris Bettencourt/Restriction Enzyme

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Achievements : Construction of 4 biobricks [Read more]: [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [Read more] Caracterisation of 2 biobricks from TUDelft [Read more]: [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site Currently we are in process of L-rhamnose-inducible promoter (pRha) caracterisation. [Read more]

Contents 1 Overview 2 Objectives 3 Design 4 Experiments and results 4.1 Mesuring efficiency of I-SceI (Cloned parts) 4.1.1 Experimental setup 4.1.1.1 Transformation results 4.1.1.2 Control for plasmid compatibility 4.1.1.3 Recovery in glucose 4.1.2 Results 4.2 Mesuring of I-SceI efficiency (TUDelft parts) 4.2.1 Experimental setup 4.2.2 Results 4.3 Characterisation of pRha 4.3.1 Experimental setup 4.3.2 Results 5 References

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

Accoring our two first objectives we should find appropriate restriction enzymes with known DNA sequence and tohoose strong promoters to regulate its expression.

Restriction ensime candidates:

1. Fse I is 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 is an intron-encoded endonuclease. It is present in the mitochondria of Saccharomyces cerevisiae and recognises an 18-base pair sequence 5'-TAGGGATAA▽CAGGGTAAT-3' (3'-ATCCC△TATTGTCCCATTA-5') and leaves a 4 base pair 3' hydroxyl overhang. It is a rare cutting endonuclease. Statistically an 18-bp sequence will occur once in every 6.9*1010 base pairs or once in 20 human genomes.

Promoter candidates:

1. pLac
   We use a standard pLac promoter from Parts Regestery: []
2. pBad
3. 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 E. coli rhaBRS locus. In the presence of Lrhamnose, RhaR activates transcription of rhaR and rhaS, resulting in an accumulation of RhaS. RhaS then acts as the L-rhamnose-dependent positive regulator of the rhaB promoter.
   • 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 to use it in our experiments:

pBad & RBS & I-SceI	pRha & RBS & GFP	pBad & RBS & RFP
pLac & RBS & I-SceI	pRha & RBS & GFP	pLac & RBS & RFP
Rha & RBS & I-SceI	pRha & RBS & GFP	pRha & RBS & RFP

Experiments and results

Mesuring efficiency of I-SceI (Cloned 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:

1. First plasmid:
2. High copy plasmid with encoded I-SceI restriction site, [http://partsregistry.org/Part:BBa_K175027 BBa_K175027]. Note: This biobrick was send us by TUDelf iGEM team.
   • Backbone: pSB1AK3
   • Resistance: Ampicillin and Kanamycin
   • Replication of Origin: modified pMB1 derived from pUC19
   • Copy number: 100-300 per cell

As result we expected to select colonies with both plasmid and

Transformation results

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

From the experiment we can clearly see that on plats with two antibiotics (Chloramphenicol & Ampicillin) there is no colonies, while on plates with only one antibiotic (Chloramphenicol or Ampicillin) there are numerous colonies. It was suggested two hipothesis to explain results:

1. Two plasmids are not compatibles. Plasmids could have different origins of replication. That is why double transformation is unsuccessful.
2. Our system works. Our system perfectly works, but there is some leakage expression of I-SceI meganuclease. In such case, it cuts I-SceI restriction site thus digest the second plasmid with ampicillin resistance.

Firstely, we decided to check the first hipothesis, and to check if two plasmids are compatible with each other.

Control for plasmid compatibility

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

Recovery in glucose

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

Results

Present your results

Mesuring of I-SceI efficiency (TUDelft parts)

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]

Experimental setup

Describe the experiment

Selection: Chloramphenicol (no induced) First plasmid: pLac & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]	Selection: Chloramphenicol (IPTG induced) First plasmid: pLac & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]
Selection: Chloramphenicol (no induced) First plasmid: pLac & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]	Selection: Chloramphenicol (IPTG induced) First plasmid: pLac & RBS & I-SceI [Cm] Second plasmid: I-SceI restriction site [Amp]

Results

Present your results

Characterisation of pRha

Experimental setup

Describe the experiment

Results

Present your results

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)