Team:Hawaii/Notebook/2008-09-18

= Things we did today =

Plasmid prep

 * Grace


 * Inoculated TB+sp100 with BB-pRL1383a for plasmid prep

PCR to verify plasmid preps

 * Grace


 * PCR of 3A:nrsg and 3A:nrpg13-8 plasmids

Construction of Secretion Device

 * Krystle


 * 3A ligation of cut pSB1A3 with the following inserts:
 * nir + gfpf + tt #8
 * nir + gfpf + tt #17
 * nir + gfpf + tt #21
 * slr + gfpf + tt #8
 * slr + gfpf + tt #17
 * slr + gfpf + tt #21

Restreaked BB-pRL1383a plate

 * Grace

Transformation
Margaret
 * transformed ligation products in DH5-alpha

Sequencing Analysis

 * Grace


 * Correct:
 * C0012V (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. Insert aligns to sequence for lacI (C0012 insert, vector was uncut)
 * C0012V-SAP'd (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. Insert aligns to sequence for lacI (C0012 insert, vector was uncut)
 * slr1+GFPf
 * Incorrect:
 * 3A:nrp: EcoRI/PstI sites present. 82bp insert aligns to sequence for E. coli flagellar biosynthesis RNA polymerase sigma factor
 * 3A:nrpg12-2: EcoRI/PstI sites present. 759bp insert aligns to sequence for E. coli fumarate reductase (anaerobic) catalytic and NAD/flavoprotein subunit
 * 3A:nrpg12-6: EcoRI/PstI sites present. 899bp insert aligns to sequence for E. coli protein C5 component of RNAse P
 * 3A:nrs2: EcoRI/PstI sites present. 85bp insert aligns to sequence for E. coli diaminopimelate decarboxylase, PLP-binding
 * 3A:nrs3: SpeI/PstI sites present. Insert aligns to pilA sequence and pSB1A3 transcriptional termination stem loop
 * 3A:nrsg10: SpeI/PstI sites present. Insert aligns to pilA + GFPf
 * pilA+GFPf12: GFPf only
 * plac+rbs1 (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. rbs (B0034) only
 * plac+rbs2 (sequence to see what it was): EcoRI/XbaI/SpeI sites present. Insert showed 97% similarity to RP4 origin of replication
 * plac+rbs12 (sequence to see what it was): EcoRI/XbaI/SpeI/PstI sites present. Insert is GFP only.
 * Resequence:
 * plac+rbs (no read -- only 5bp sequenced ?!?)
 * pilA+GFPf13 (no read -- only 5bp sequenced ?!?)

= Discussion =
 * Please throw away old plates in 4C fridge. Some have fungi growing on them already.

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