Revision as of 21:58, 7 September 2008

7th September 2008

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
- Monday or Tuesday:PCR trial all genomicseqeunces to determineoptimal conditions with Taq

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

@@ Line 46: / Line 46: @@
 *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-Blue''E.coli''