Team:Hawaii/Notebook/2008-10- 6

= Things we did today =

Construction of secretion device (cont.)

 * Grace


 * PCR'd nir, nir+rbs, rbs+GFP+tt#2, plac+rbs, slr1+GFPf
 * Verified PCR products on 0.8% agarose gel
 * Overnight RE digest:
 * EcoRI/SpeI:
 * nir
 * nir+rbs
 * plac+rbs
 * XbaI/PstI;
 * slr1+GFPf
 * pilA+GFPf+tt
 * rbs+GFP+tt#2
 * HindIII/BamHI:
 * J33207

OriV construction

 * Margaret
 * Ligation
 * oriV + pSB1A3 (de-phosphorylated)


 * Transformation into DH5-alpha, with amp100 selection
 * 10/4 ligation
 * 10/6 ligation
 * (+) control pUC18
 * (-) control no plasmid


 * PCR verification
 * colonies 5 and 6 were PCR verified
 * a gel was run
 * oriV1---> band at 400 bp
 * oriV2---> no band
 * oriV3---> no band
 * oriV4---> no band
 * oriV5---> band at 700 bp
 * oriV6---> band at 1000 bp
 * rep 2---> band at 3000 bp
 * plac/rbs/rep (colony 8)---> band <3kb (need to re-do this)

plac/rbs/rep and plac/rbs/aada

 * Margaret


 * plasmid prep of both constructs
 * Restriction digest for 2 hours at 37&deg;C, then a gel was run to verify the digest
 * rep2 with X,P
 * plac/rbs/rep (colony 8) with X,P
 * plac/rbs/rep (colony 8) with E,S
 * plac/rbs/aada with X,P
 * PCR verification of more plac/rbs/rep colonies (14 total) and also colony 8 again to verify that I have what I think I do. (+) was pSB1A3, (-) was water

Sequencing analysis

 * Grace


 * Correct:
 * rbs+GFP+tt #1, 2
 * BBpRL1383a:
 * All four sequenced colonies had a G->C single bp substitution at position 229 of part (in CAP binding site)
 * Colony 18 confirmed (best quality sequence)
 * Colony 1 confirmed (abiguities at 225, 583, 591, 552 of part)
 * Colony 3 (abiguities at 530, 513 of part)
 * Colony 13 (low quality sequence, but BioBrick MCS is present)
 * Incorrect:
 * nir+rbs+GFP #4, 7
 * nir+rbs+slr1+GFPf #8
 * No sequence obtained:
 * plac+rbs
 * nir+rbs+GFP #1
 * nir+rbs+slr1+GFPf #2
 * nir+rbs+pilA #17

= Discussion =

= Quote of the Day = "History is the only laboratory we have in which to test the consequences of thought. - Étienne Gilson"