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Team:Stanford-Brown/Protocols
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=== '''Venus Life''' === | === '''Venus Life''' === | ||
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| + | '''Construct Development''' | ||
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| + | Potential cell-cycle dependent promoters in E. coli and their sequences were identified from literature (Quiñones 1997, Sun 1994, Ogawa 1994). Primers were designed to isolate these sequences and add on BioBrick cut sites according to the Silver lab method (Phillips 2006). Colonies of E. coli were selected from plates and sequences were isolated using colony PCR. Sequences were verified using BioPharm oligo sequencing and gel electrophoresis. PCR product was digested for 2 hours at 37C with EcoRI and SpeI. Digestion products were isolated via gel extraction; two samples were ligated with pSB1C3 cut with EcoRI and SpeI, and BBa_E0840 cut with EcoRI and XbaI, respectively. Ligation products were transformed into NEB-5a competent cells via heat-shock; promoter-pSB1C3 strains were plated then inoculated in LB+Chloramphenicol and promoter-E0840 strains were plated then inoculated in LB+Ampicillin. | ||
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| + | '''Bulk Assay''' | ||
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| + | 50mL cultures of promoter-E0840 were grown overnight in 125mL flasks with LB+Amp, and some culture was diluted into 2 15mL samples with OD600 between 0.1 and 0.3. To synchronize cell cycle, serine hydroxamate (SHX) was added to both replicates to a final concentration of 10mg/mL and allowed to incubate at 37C for 1.5 hours (Ferullo 2009). After incubation, OD600 was taken to verify cell-cycle arrest. Samples were pelleted and resuspended in LB+Amp and replaced in the 37C shaking incubator. | ||
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| + | Every 2.5 minutes, 1mL sample was removed from each replicate and OD600 was measured. The same sample was transferred into a microcentrifuge tube, pelleted, aspirated, and resuspended in M9. This sample was then measured in the fluorometer. | ||
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| + | '''Microscopy Assay''' | ||
| + | |||
| + | 4mL cultures of promoter-E0840 were grown overnight in 15mL conicals with LB+Amp. At an OD600 reading between 01. and 0.3, 40uL of 100X SHX was added to synchronize cell cycle. After 90 minute incubation, cells were pelleted and resuspended in LB+Amp without serine hydroxamate. 10uL of sample was pipetted onto a glass slide then covered with an agar pad. A timelapse was taken for 2 hours, taking images every 5 minutes. | ||
=== '''Biomining''' === | === '''Biomining''' === | ||
Revision as of 02:58, 3 October 2012
Contents |
Methods
General
Hell Cell
Venus Life
Construct Development
Potential cell-cycle dependent promoters in E. coli and their sequences were identified from literature (Quiñones 1997, Sun 1994, Ogawa 1994). Primers were designed to isolate these sequences and add on BioBrick cut sites according to the Silver lab method (Phillips 2006). Colonies of E. coli were selected from plates and sequences were isolated using colony PCR. Sequences were verified using BioPharm oligo sequencing and gel electrophoresis. PCR product was digested for 2 hours at 37C with EcoRI and SpeI. Digestion products were isolated via gel extraction; two samples were ligated with pSB1C3 cut with EcoRI and SpeI, and BBa_E0840 cut with EcoRI and XbaI, respectively. Ligation products were transformed into NEB-5a competent cells via heat-shock; promoter-pSB1C3 strains were plated then inoculated in LB+Chloramphenicol and promoter-E0840 strains were plated then inoculated in LB+Ampicillin.
Bulk Assay
50mL cultures of promoter-E0840 were grown overnight in 125mL flasks with LB+Amp, and some culture was diluted into 2 15mL samples with OD600 between 0.1 and 0.3. To synchronize cell cycle, serine hydroxamate (SHX) was added to both replicates to a final concentration of 10mg/mL and allowed to incubate at 37C for 1.5 hours (Ferullo 2009). After incubation, OD600 was taken to verify cell-cycle arrest. Samples were pelleted and resuspended in LB+Amp and replaced in the 37C shaking incubator.
Every 2.5 minutes, 1mL sample was removed from each replicate and OD600 was measured. The same sample was transferred into a microcentrifuge tube, pelleted, aspirated, and resuspended in M9. This sample was then measured in the fluorometer.
Microscopy Assay
4mL cultures of promoter-E0840 were grown overnight in 15mL conicals with LB+Amp. At an OD600 reading between 01. and 0.3, 40uL of 100X SHX was added to synchronize cell cycle. After 90 minute incubation, cells were pelleted and resuspended in LB+Amp without serine hydroxamate. 10uL of sample was pipetted onto a glass slide then covered with an agar pad. A timelapse was taken for 2 hours, taking images every 5 minutes.
