Revision as of 23:15, 7 September 2008 by Qiqin (Talk | contribs)

7th September 2008

Wetlab

PCR trial all genomic sequences to determine optimal conditions with Taq then produce clones with Pfu
Digest with XbaI and SpeI and incorporate into biobricks
Follow up by assaying orientation (either with sequence internal restriction site or by using XbaI and SpeI, as they should only be able to cut inserts in the correct orientation)

GeneArt constructs 7, 8, 9 and 11 (SacB-EAK16, LipA-EAK16, P43-RBSgsiB and Pveg-RBSgsiB) to be ligated into Biobrick vector pSB1A2 or pSB1AK3
CAT to be ligated togethre with the terminator (B0015) to form CAT-Terminator
AmyE 5' and 3' integration sequences and Aad9 (Spectinomycin resistance gene) to be integrated into pSB1A2 or pSB1AK3
GeneArt constructs 3, 6, 10 and 12 (EpsE gene and PgsiB-RBSspoVG in construct 3, LipA-Elastin and PgsiB-RBSgsiB in construct 6, P43-RBSspoVG and Pveg-RBSspoVG), along with all above biobricks will be transformed into Xl1-Blue E.coli
Depending on results, LacI PCR to be digested and purified ready for ligation into pSB1A2/pSB1AK3

E.coli containing GeneArt Constructs 7, 8, 9, 11 and CAT-Terminator to be grown up for midi-prepping Wednesdays
E.coli containing AmyE 5' and 3' integration sequences and Aad9 to be grown for mini-prep on Wednesday
GFP-Terminator to be cut ready for building
Ligate LacI into biobrick and transform into XL1-Blue

Constructs 7, 8, 9, 11 and CAT-Terminator to be midi-prepped and digested ready for building of the phase 1 constructs
E.coli containing constructs 10 and 12 to be grown up overnight for midi-prepping
Aad9 and the AmyE integration sequences to be miniprepped and digested to check insert orientation
Correctly orientated biobricks to be grown up overnight for midi-prepping Thursday
Grow E.coli containing the new LacI biobrick
Time permitting (If not will be carried out Thursday):
- CAT-Terminator to be ligated to P43-RBSgsiB (9) and Pveg-RBSgsiB (11) separately OR AmyE 3' integration sequence tomorrow (depends on status of AmyE 3' sequence) and transformed into XL1-BlueE.coli
- GFP-Terminator to be ligated to P43-RBSgsiB (9) and Pveg-RBSgsiB (11) separately and transformed into XL1-BlueE.coli(depending on CAT-Terminator ligation petner)

Constructs 10, 12 the AmyE integration sequences and Aad9 to be midiprepped
AmyE integration sequences and Aad9 to be digested ready for builiding of phase 1 construct
Constructs 10 and 12 to be digested and gel-purified ready to be incorporated into biobricks
E.coli containing constructs 3 and 6 to be grown up overnight for midi-prepping
Mini-prep the LacI biobrick and check for correct orientation. Grow a culture with the LacI gene in the correct orientation for midi-prep tomorrow
Time permitting (If not may be carried out Friday):
- Aad9 to be ligated to the P43-RBSgsiB and Pveg-RBSgsiB separately and transformed into XL1-BlueE.coli
- Terminator to be ligated AmyE integration sequeces (separately) and transformed into XL1-BlueE.coli
- Constructs 10 and 12 to be ligated into biobricks

Constructs 3 and 6 to be midiprepped
Constructs 3 and 6 digested and run on a gel to separate components by gel purification
Midi-prep LacI
Finish any unfinished jobs from earlier in week

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