Team:Brown-Stanford/Lab/Notebook/Week5
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+ | == ''' July 11, 2011''' == | ||
+ | |||
+ | ===PowerCell=== | ||
+ | *Transformation of GFPmut3B into TOP10 went okay, | ||
+ | *Made liquid culture in LB + amp (100 μg/ml) | ||
+ | *4ml BG11, 1ml from stock culture | ||
+ | *Made more working stocks of cyanos | ||
+ | *Rehydrated CscB into 20ul TE; transformed TOP10 cells; PCR with Gibson primers (two PCRs: RBS|CscB, CscB|TTSP) | ||
+ | *transformed 100ul TOP10 cells with 2ul of hydrated CscB; x2 replicates; 25min on ice, 2min heat shock @ 42C, then add 500ul LB and place in 37C for ~20min | ||
+ | *two plates in 37C at 5:30pm | ||
+ | *Viewed plates at 9:30am, 100s of colonies; placed in 4C in Rm335 | ||
+ | |||
+ | *Make antibiotic plates (jov/lei), | ||
+ | *Kanamycin 40 ug/mL | ||
+ | *ampicillin 100 ug/mL | ||
+ | *tetracycline 10 ug/mL | ||
+ | *need amp/tet, amp, kan | ||
+ | *Design primers for veg promoter + GFP-only constructs | ||
+ | *PCR amplfication anabaena vegetative promoter-mid RBS | ||
+ | *PCR cleanup on Nostoc promoter--resulted in 25.8ng/ul of dna product, some fifteen μl | ||
+ | *micrographs using oil immersion, get condenser-optimized pictures of cyanos | ||
+ | *(*synechocystis 6803: need real picture of cells; cyanothece 7822 needs denser sample; anabaena 7120 needs non-airbubble) | ||
+ | == ''' July 12, 2011''' == | ||
+ | |||
+ | ===PowerCell=== | ||
+ | *work division: Eli, Julius, Evan, yet to do, done | ||
+ | *imaged PCRs from 7/11: two cscB rxns (the RBS-cscB part, and the cscB-TTSP part); Anabaena promoter-mid PCR as well | ||
+ | *miniprep’d the GFPmut3b out of transformant TOP10 liquid cultures. Plasmids kept in tray with norman’s plasmids in 335 4?C. | ||
+ | *streaked out Elhai and Norman plasmids | ||
+ | *Norman plasmids: 100ug/ml for pRL1383a | ||
+ | *cscB liquid culture in preparation for plasmid harvest on 7/13 | ||
+ | *innoculated 2ml of LB amp, 3x replicates; placed in 37C at 5:15pm | ||
+ | *finished cryostock | ||
+ | *PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends | ||
+ | == ''' July 12, 2011''' == | ||
+ | ===PowerCell=== | ||
+ | Listened to Jay Keasling's speech on applying synbio to growing biological organisms. | ||
+ | == ''' July 13, 2011''' == | ||
+ | |||
+ | ===PowerCell=== | ||
+ | *image PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends. not much showed up, although bands were present in last lanes (identity?) | ||
+ | *cut out most visible (Ana Strong) for gel extraction. | ||
+ | *plasmid prep cscB liquid cultures from 7/12 | ||
+ | *picked colony from conjugal transformation plasmid lines and make antibiotic liquid cultures | ||
+ | ===REGOBrickes=== | ||
+ | *Made new Bang plates, and resolved the issue with pH. Instead of pHing to 6.8, pHed to 8.5, which helped growth considerably. | ||
+ | *Possible plasmids for transformation of S. pasteurii (from B subtilis protocols) | ||
+ | **pUB110 | ||
+ | **pHT43 | ||
+ | **pHT 01 | ||
+ | *Others, from Keggins et al (uploaded) include | ||
+ | **pPL576 | ||
+ | **pPL10 | ||
+ | **pPL7065 | ||
+ | **pPL2 | ||
+ | == ''' July 14, 2011''' == | ||
+ | |||
+ | ===PowerCell=== | ||
+ | *cryostock cargo/helper/conjugative plasmid strain liquid cultures | ||
+ | *gel extraction on Ana strong band cut on 7/13 (nanodrop: <5ng/ml) | ||
+ | *imaged Ana weak, Nos weak/strong (from 7/12 PCR) | ||
+ | *cut out 600bp band of Ana weak 50- (there were two bands, strong @ ~600bp for 50-, very faint around 500bp for all) and ~600bp band for Nos weak (all four temps), gel extraction | ||
+ | *plasmid prep of liquid conjugal transformation plasmid cultures (just the ones we will eventually need to modify); stored @4C in Rm 335, “Norman’s plasmids” rack | ||
+ | *pRL25: 30.4ng/ul | ||
+ | *pRL1383a: 26.4ng/ul | ||
+ | *BBpRL: 16.75ng/ul | ||
+ | *ran big PCR of cscB with two ends, cscB with wk and str RBS’s needed for gibson | ||
+ | |||
+ | ===REGOBricks=== | ||
+ | *printed out research articles detailing transformation protocols for subtilis that we hope to adapt for pasteurii: | ||
+ | *Digestion to Protoplast, then transformation | ||
+ | *soil transformation | ||
+ | *CaCl<sub>2 </sub> | ||
+ | |||
+ | *features/reasons to use pUB110: | ||
+ | **EcoR1 cut sites | ||
+ | **50 copies/cell | ||
+ | *If we needed to try phage transformation, pUB was a lot more amenable to those protocols than other plamsids. | ||
+ | *Other vectors include two from Cambridge (2008): | ||
+ | *pBSINT1 - B. subtilis integration vector (5632 bp) - inserted in amylase gene | ||
+ | *pBSEP - episomal | ||
+ | == ''' July 15, 2011''' == | ||
+ | |||
+ | ===PowerCell=== | ||
+ | *imaged PCR of cscB with two ends, cscB with wk and str RBS’s (from 7/14) | ||
+ | *visible bands for cscB-TTSP, RBSw cscB and RBSs cscB; cut out bands and ran gel extraction | ||
+ | *imaged Ana mid, positive control and negative control for RBSm-cscB (from PCR done on 7/11) | ||
+ | *cut out Ana med bands, ran gel extraction; also performed PCR cleanup using Qiagen kit (result 5ng/ul for gel ext, 17.5ng/ul for PCR cleanup) to use for 2nd round PCR | ||
+ | *imaged RBS-GFP-TTSP from 7/12, (-) cont for GFP from 7/14; | ||
+ | *cut out four bright bands, NOT YET GEL EXTRACTED; also enough quantity to PCR cleanup directly (NOT YET DONE) | ||
+ | *PCR: second round Ana weak, strong, med, Nostoc weak | ||
+ | *PCR: RBSm|cscB | ||
+ | ===REGOBricks=== | ||
+ | *Dosier Media | ||
+ | **5g soy protein | ||
+ | **15g peptone from casein | ||
+ | **10 micrograms manganese sulfate | ||
+ | **20 g urea | ||
+ | **5g NaCl | ||
Revision as of 20:17, 25 September 2011
July 11, 2011
PowerCell
- Transformation of GFPmut3B into TOP10 went okay,
- Made liquid culture in LB + amp (100 μg/ml)
- 4ml BG11, 1ml from stock culture
- Made more working stocks of cyanos
- Rehydrated CscB into 20ul TE; transformed TOP10 cells; PCR with Gibson primers (two PCRs: RBS|CscB, CscB|TTSP)
- transformed 100ul TOP10 cells with 2ul of hydrated CscB; x2 replicates; 25min on ice, 2min heat shock @ 42C, then add 500ul LB and place in 37C for ~20min
- two plates in 37C at 5:30pm
- Viewed plates at 9:30am, 100s of colonies; placed in 4C in Rm335
- Make antibiotic plates (jov/lei),
- Kanamycin 40 ug/mL
- ampicillin 100 ug/mL
- tetracycline 10 ug/mL
- need amp/tet, amp, kan
- Design primers for veg promoter + GFP-only constructs
- PCR amplfication anabaena vegetative promoter-mid RBS
- PCR cleanup on Nostoc promoter--resulted in 25.8ng/ul of dna product, some fifteen μl
- micrographs using oil immersion, get condenser-optimized pictures of cyanos
- (*synechocystis 6803: need real picture of cells; cyanothece 7822 needs denser sample; anabaena 7120 needs non-airbubble)
July 12, 2011
PowerCell
- work division: Eli, Julius, Evan, yet to do, done
- imaged PCRs from 7/11: two cscB rxns (the RBS-cscB part, and the cscB-TTSP part); Anabaena promoter-mid PCR as well
- miniprep’d the GFPmut3b out of transformant TOP10 liquid cultures. Plasmids kept in tray with norman’s plasmids in 335 4?C.
- streaked out Elhai and Norman plasmids
- Norman plasmids: 100ug/ml for pRL1383a
- cscB liquid culture in preparation for plasmid harvest on 7/13
- innoculated 2ml of LB amp, 3x replicates; placed in 37C at 5:15pm
- finished cryostock
- PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends
July 12, 2011
PowerCell
Listened to Jay Keasling's speech on applying synbio to growing biological organisms.
July 13, 2011
PowerCell
- image PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends. not much showed up, although bands were present in last lanes (identity?)
- cut out most visible (Ana Strong) for gel extraction.
- plasmid prep cscB liquid cultures from 7/12
- picked colony from conjugal transformation plasmid lines and make antibiotic liquid cultures
REGOBrickes
- Made new Bang plates, and resolved the issue with pH. Instead of pHing to 6.8, pHed to 8.5, which helped growth considerably.
- Possible plasmids for transformation of S. pasteurii (from B subtilis protocols)
- pUB110
- pHT43
- pHT 01
- Others, from Keggins et al (uploaded) include
- pPL576
- pPL10
- pPL7065
- pPL2
July 14, 2011
PowerCell
- cryostock cargo/helper/conjugative plasmid strain liquid cultures
- gel extraction on Ana strong band cut on 7/13 (nanodrop: <5ng/ml)
- imaged Ana weak, Nos weak/strong (from 7/12 PCR)
- cut out 600bp band of Ana weak 50- (there were two bands, strong @ ~600bp for 50-, very faint around 500bp for all) and ~600bp band for Nos weak (all four temps), gel extraction
- plasmid prep of liquid conjugal transformation plasmid cultures (just the ones we will eventually need to modify); stored @4C in Rm 335, “Norman’s plasmids” rack
- pRL25: 30.4ng/ul
- pRL1383a: 26.4ng/ul
- BBpRL: 16.75ng/ul
- ran big PCR of cscB with two ends, cscB with wk and str RBS’s needed for gibson
REGOBricks
- printed out research articles detailing transformation protocols for subtilis that we hope to adapt for pasteurii:
- Digestion to Protoplast, then transformation
- soil transformation
- CaCl2
- features/reasons to use pUB110:
- EcoR1 cut sites
- 50 copies/cell
- If we needed to try phage transformation, pUB was a lot more amenable to those protocols than other plamsids.
- Other vectors include two from Cambridge (2008):
- pBSINT1 - B. subtilis integration vector (5632 bp) - inserted in amylase gene
- pBSEP - episomal
July 15, 2011
PowerCell
- imaged PCR of cscB with two ends, cscB with wk and str RBS’s (from 7/14)
- visible bands for cscB-TTSP, RBSw cscB and RBSs cscB; cut out bands and ran gel extraction
- imaged Ana mid, positive control and negative control for RBSm-cscB (from PCR done on 7/11)
- cut out Ana med bands, ran gel extraction; also performed PCR cleanup using Qiagen kit (result 5ng/ul for gel ext, 17.5ng/ul for PCR cleanup) to use for 2nd round PCR
- imaged RBS-GFP-TTSP from 7/12, (-) cont for GFP from 7/14;
- cut out four bright bands, NOT YET GEL EXTRACTED; also enough quantity to PCR cleanup directly (NOT YET DONE)
- PCR: second round Ana weak, strong, med, Nostoc weak
- PCR: RBSm|cscB
REGOBricks
- Dosier Media
- 5g soy protein
- 15g peptone from casein
- 10 micrograms manganese sulfate
- 20 g urea
- 5g NaCl