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Team:EPF-Lausanne/Notebook/Cloning Strategy
From 2009.igem.org
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<br>SpeI ACTAGT | <br>SpeI ACTAGT | ||
<br>PsiI TTATAA | <br>PsiI TTATAA | ||
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| + | Design the primers for the 2 step-PCR: | ||
| + | the first step introduces the LacI promoter and the RBS upstream the LovTAP gene with the Forward primer, whereas the Reverse introduces the Term downstream. | ||
| + | In the second step we only introduce the E-X restriction sites upstream and the SP downstream. | ||
===22.07.09=== | ===22.07.09=== | ||
Revision as of 14:32, 21 July 2009
Contents |
Cloning strategy
July
06.07.09
Four forward primers were designed to amplify:
1.Promoter T7, RBS, CBP and LOVTAP:
- gtttcttcgaattcgcggccgcttctagagtaatacgactcactataggggaattgtg
2.RBS, CBP and LOVTAP:
- gtttcttcgaattcgcggccgcttctagagtgtttaactttaagaaggag
3.CBP and LOVTAP:
- gtttcttcgaattcgcggccgcttctagatgaagcgacgatggaaaaagaatttcatag
4.LOVTAP:
- gtttcttcgaattcgcggccgcttctagatgctactacacttgaacgtattgagaagaac
One reverse primer were designed:
- gtttcttcctgcagcggccgctactagtatcaatcgcttttcagcaacacctcttc
The recipient IGEM part have been chosen: [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_B0010 BBa_B0010], well 13D in the received kit plate 1
07.07.09
To design plasmids : software Vector NTI
08.07.09
Inducible LOVTAP biobrick strategy
- Problem to overcome:
- PstI sites in LOVTAP sequence.
- Our goal:
- Biobrick consisted of LacI promoter-RBS-LOVTAP-Term (in this order).
- Material:
- Biobrick of LacI promoter, RBS, LOVTAP, Term separately ( LOVTAP obtained from previous section and the rest from iGEM Spring 2009 distribution Kit plate).
- Strategy:
- LacI promoter-RBS ligation with iGEM protocol (LacI promoter digested with ES, RBS digested with XP, plasmid containing an other antibiotic digested with EP).
- LOVTAP is digested with ES and inserted into Term plasmid (which was digested with EX previously).
- Finally, LacI promoter-RBS digested with ES to be inserted into LOVTAP-Term plasmid, digested with EX.
09.07.09
Partial digestion strategy
- Problem to overcome:
- PstI sites in LOVTAP sequence -> Impossible to use iGEM protocol for ligation (where downstream part must be cut with XP)
- Strategy:
- Cut with X first.
- Divide into several solutions and add different concentrations of P (by dilution series)
- Results:
- Most concentrated tube: all P sites are recognized and cut, so the number of parts diminishes with the concentration value.
- Run an agarose gel and extract the right piece (recognized by the segment's length).
10.07.09
13.07.09
Restriction enzymes on [http://www.neb.com/nebecomm/products/category1.asp?#2 Biolabs website] and [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp clevage oligonucleotides]
TRP promoter biobrick strategy
- Problem to overcome:
- SpeI sites on Trp promoter sequence and it's an upstream part which has to be cut with ES.
- Strategy:
- PCR: Forward primer having E and X sites and Reverse primer NheI.
- Digest Trp promoter with E and NheI.
- Digest plasmid with E and X.
- Ligation -> E site is recreated; X and NheI have compatible ends so ligation is possible and the site is destroyed (mixted site).
14.07.09
Primers designed for LOVTAP read-out and RBphP project.
15.07.09
16.07.09
17.07.09
20.07.09
21.07.09
One useful website to know the restriction sites of enzymes:
http://www.genscript.com/cgi-bin/products/enzyme.cgi?op=all_ez
The restriction site used were:
EcoRI GAATTC
XbaI TCTAGA
SpeI ACTAGT
PsiI TTATAA
Design the primers for the 2 step-PCR: the first step introduces the LacI promoter and the RBS upstream the LovTAP gene with the Forward primer, whereas the Reverse introduces the Term downstream. In the second step we only introduce the E-X restriction sites upstream and the SP downstream.
