Learned how to make competent cells, growing up two strains for tomorrow
Transformed 8 plasmids
Determined EL222 fusion is risky but still going ahead with it
Linkers are totally setlled
Found zinc finger plasmid and updated target sequence
Learned how to make tetr- mcherry fusion
Settled on 5 promoters
5-Jun
Learned how to make competent cells, testing them and then making more tomorrow
Transformed 8 plasmids again
Made primers to clone the TET-GFP reporter system, the mCherry promoter strength system,
ready to order
Made ultramers for variable promoter blocks (and no target neg controls) – ready to order
Spilled a lot of iced tea outside, bummer
Started primers for dna binding machines
Got a handle on cas9 fusions (pun intended).
Put awesome pics in dropbox
6-Jun
Clean up dropbox
Update budget sheet with addgene and cell center orders
Finish primers for fusion
Set up plate reader for GFP and mCherry assays
run minipreps on pdawn, pdawn-mcherry, pet26b
Grow up mCherry stock
Wrote Penn iGEM on our plasmid
7-Jun
Transform
C0012 –amp/chlor (do both)
M11307 – amp/chlor (do both)
I13458 – amp/chlor (do both)
R0010 – amp/chlor (do both)
R0051 – amp
K206000 –chlor
Start the LIMS and file all the strains and DNA we have made/ ordered
8-Jun
Miniprep Addgene stuff + transformations that worked
Growing up low copy plasmids in 40mLs
Mini-prepped
I9002
I13458
C0051
Pdawn-mcherry
Pdawn
Dhsa mcherry
Pdawn dhsa
Psb1a3
JM mcherry
Pet26b
WEEK 2
June 11 2013 - June 18 2013
17-Jun
Grow up luxI culture and grow up tetR culture
Sequence all of the minipreps
Transform t9002 in psb1A3 in NEB10
Retransform ptetGFP to see if BL21DE3 cells are competent
Transform r0079, k081015, r0063 in NEB10
Miniprep psb1k3
Redo dam gel with more dna
Figure out second control zfp from addgene
Figure out how to add luxR binding site to target region
Order sequencing primers for all addgene minipreps
Bisulfite converted msssi methylated c0051
18-Jun
The bisulfite conversion was missing a negative control (bisulfite converted but unmethylated c0051) we'll need this to interperet results
Miniprep c0078, c0079 + make glycerol stocks check same plasmid with our kit
Order 13420 (second zfp)
Design a way to make variable promoters more easily varied (for a biobrick so teams can use the reporter plasmid for their own nefarious reasons)
19-Jun
Transform failed transformation
Make competent DH5a && Dam-
Figure out methylation assays for promoters
Miniprep psb1A3 && all the 40mL cultures
Picked many colonies
Check pTet-gfp under blue light
WEEK 3
June 18 2013 - June 25 2013
21-Jun
troubleshoot plux-luxI pcr
roubleshoot pdawn-luxI pcr
made pDawn-tetR pcr work
troubleshoot pet26b-tetR pcr
troubleshoot pDawn-GFP pcr
troubleshoot pDawn-mCherry-secretion tag pcr
miniprep growing cultures, be sure to pick only the glowing ligations
ransform the correct t9002 amp ligation - determined from gel
digested t9002 in amp and ptet gfp in amp to identify the correct ligation
all of the chosen ptet gfp ligations worked, but let's not use 5 || 13 12. troubleshoot t9002 digest
troubleshoot t9002 digest
check for contamination of something (run uncut sample, sample + buffer, sample + 1
enzyme, sample + other enzyme, sample + both enzymes)
24-Jun
Dam-/dh5a v dam methylation + dpnI && dpnII digest
Digested/ligated/transformed t9002 in amp
Get methylated biobrick sequenced
get chlor backbones sequenced
culture t9002 transformations in liquid media with i751250
mini prep stuff in the incubator
figure out the primer issues 8
Pick t9002 colonies for miniprep
WEEK 4
June 25 2013 - July 2 2013
26-Jun
Dam-/dh5a v dam methylation + dpnI && dpnII digest
Digested/ligated/transformed t9002 in amp
get chlor backbones sequenced
culture t9002 transformations in liquid media with i751250
mini prep stuff in the incubator
figure out the primer issues
Pick t9002 colonies for miniprep
USER Cloning reporter plasmid
1-Jul
Beautiful Brady Bunch photoshoot
Troubleshooted and Re-tred PCR for user ends for reporter plasmid
Called IDT about pcr assembly – they said Gibson tends to work better, no mutations, all in one tub. If we must PCR assembly – add DMSO, hotstart reaction, anneal at 68-70C (we did this).
Get methylated biobrick sequenced
Only sequence ptet GFP 11, if verified make sure to note on LIMS that we are only using ptet GFP 11
Check if plux/luxI system is working in liquid cultures – this failed
a. Might be strain competition, need to know growth rates
Re-suspend primers for lux amplifier
Mini-prep: e0040, psb1a3, r0062
2-Jul
Think about application of mathylation project in e.coli
Ceck if plux/GFP-psb1C3 system is working in liquid cultures
+/- AHL induction at 100nM
Compare with ptetGFP fluorescence, normal LB fluorescence
Streak zinc finger 2
Grow up 44251
Transform up R0062
When BstuI arrives
Assay BstuI working
Assay the bvluc and tale1 minipreps in dh5a, dam-, and bl21 cells with msssi and bstuI
Results: BstUI is blocked by methylation, and cells don’t normally methylate
Growing up t9002 in chlor and i751250 in amp for fluorescence study
Investigate CHIP or other ways of determining DNA binding domain specificity
WEEK 5
July 2 2013 - July 9 2013
3-Jul
Streak zinc finger 2
Grow up 44251
Look into lux box being light sensitive
4-Jul
Mini prep 44251
Pick ZFP2 colonies to grow up, throw out liquid culture in fridge
5-Jul
Repeat BstUI assay, taking into account new controls
Suspend the primers in the freezer
We need to check if the origin of replications are compatible before co transformation
Characterize pDawn-mcherry
Practice measuring fluorescence
6-Jul
troubleshoot ptetGFP user PCR - band was visible but too small to extract