followed special measures for using EcoRI and SpeI with Buffer 2
2.5% enzymes by volume
but also maintaining a 10 unit: 1μg ratio of EcoRI to plasmid to avoid star activity
digestion success: verification gel has solid bands where expected
ligation
actually pulled off 10 μl reactions
included Xho1 enzyme to break up undesired lux component and hopefully increase chances of proper ligation pairings
also have enough digestion product remaining to do gel extraction if necessary
Transformed Promoterless-GFPmut3b from registry: Well 22I, Plate 1
August 30, 2011
e coli W competency & transformation w/ RFP
transformation of Ana TOPO +lux/pSB1C3 ligation w/ XhoI
transformation of cscB PCR +lux/pSB1C3 ligation w/ XhoI
redo ligations from 8/29 with HindIII instead of XhoI
turns out XhoI cuts pSB1C3
transform ligations redux
warmed plates
transformation of promoterless GFP from registry
all transformations (except GFP) performed on both TOP10 and K12
new liquid cultures of pRL443 and pRL623+25\
Made 3 amp plates. Running out of LB. hopefully we dont need more.
August 31, 2011
we have transformants all over the place!
Our TOP10 sucks compared to K12
Many colonies on the Ana-TOPO+lux/pSB1C3 HindIII K12 plate
three colonies on the cscB-PCR+lux/pSB1C3 HindIII K12 plate
one colony on the promoterless GFP TOP10 plate
a few colonies on the Ana-TOPO+lux/pSB1C3 XhoI K12 plate
a few colonies on the Ana-TOPO+lux/pSB1C3 XhoI TOP10 plate
Update Sept 1: 2 colonies cropped up on the Ana HindIII TOP10 plate!
verification PCR and gel of BioBrick transformation results
liq cultures transformations if verification looks good
verification looked... unconclusive, some things that *could* be what we want.
liquid cultured them, will miniprep, verify PCR / sequence.
AHK 2,4,5 ; CHK 2 ; GFPN ; AXT 3
cyano transformation, orgy part
agar stab ecoli W
September 1, 2011
made 1 liter LB
and BG11 (for cyano plates)
another ligation of cscB+pSB1C3(w/ lux)
to transform and get more candidate colonies
re-inoculate GFP
transfer cyanos to selective plates
2 colonies cropped up on the Ana HindIII TOP10 plate Biobrick transform plate from Aug 30! Put in the 4C in 335.
Miniprepped AHK2,4,5 ; CHK2 ; AXT3 BB candidates
Nanodrop results:
AHK2: 75 ng/ul
AHK4: 76 ng/ul
AHK5: 76ng/ul
CHK2: 186ng/ul
AXT3: 193 ng/ul
Sent the miniprepped BB cands. off for sequencing from Elim
Replated dried cyano slides/nitrocellulose control test from Aug 31 onto plain BG11 Plates
Slides - added 10ul BG11 over dried spot, scratched around, sucked up as much as pos, put on plate
nitrocellulose: just dropped on plate
September 2, 2011
colonies growing on cscB redux K12
confirmed now that TOP10 sucks, will be using K12 only from now on
liq cult of GFP looks good, ready to miniprep
completed miniprep; only 22 ng/μl but thatll do
AHK4 plasmid sequencing results came out beautifully
WE HAVE ANA PROMOTER BIOBRICK!!
AHK5 also promising but AHK4 looks like our prize. AHK5 is correct in the reverse direction, but bad read in the fwd direction. From gel they look similar, so we believe they’re both correct, but we’re only choosing AHK4 because we only need one and it’s perfect in both fwd and reverse.
CHK2 turned out to be the luxbrick re-ligated into psb1c3 even though we cut it up 4 times with HindIII! Interesting. Wonder how effective adding HindIII really is.
unknown what AXT3 is.
digestion and ligation of Ana-GFP construct
50 μl digestions
cut Ana-pSB1C3 w/ Spe1, Pst1
cut GFPN w/ Xba1, Pst1
ana ligation ended up at 15μl, but adjusted enzymes accordingly, and checked with kosuke about best approach
adjusted ligase and buffer amounts accordingly
3:1 insert to vector molar ratio
~50 ng vector (ana-pSB1C3 digest)
ligated at room temp for 2 hrs, -> -20 until Monday
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