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- week 1
- week 2
- week 3
- week 4
- week 5
WEEK 1
June 4 2013 - June 11 2013
- 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
- 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
- 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
- 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
- Miniprep Addgene stuff + transformations that worked
- Growing up low copy plasmids in 40mLs
- I9002
- I13458
- C0051
- Pdawn-mcherry
- Pdawn
- Dhsa mcherry
- Pdawn dhsa
- Psb1a3
- JM mcherry
- Pet26b
WEEK 2
June 11 2013 - June 18 2013
- 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
- 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)
- 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
- 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)
- 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
- gel extract promoter fragments from USER PCR
- re-do USER PCR for: TetR, pTetGFP
- Design/check/order (Tale,Cas,ZFP)-flex-REV primers - check fwd primers
