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Team:Groningen/Notebook/22 August 2009
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m (New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== :→ {{todo}} Isolate plasmids from E.coli Top10 BBa_J23101 cells :→ {{todo}} Plate E.coli Top10 BBa_J23101 cells from...) |
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===GVP Cluster=== | ===GVP Cluster=== | ||
| - | :→ {{ | + | :→ {{done}} Isolate plasmids from E.coli Top10 BBa_J23101 cells |
| - | :→ {{ | + | :→ {{done}} Plate E.coli Top10 BBa_J23101 cells from o.n. culture |
| - | :→ {{todo}} Pellet remaining cells for short storage in -20 box | + | :→ {{todo}} <strike>Pellet remaining cells for short storage in -20 box</strike> (not needed) |
| - | :→ {{ | + | :→ {{done}} Place the four test cultures with and without GVP on table at room temperature (make foto) |
| + | |||
| + | :→ {{done}} Look at plates stored at 37C with BBa_J61002-pArsR<sup>+</sup>/pZntR<sup>+</sup>/pCueO<sup>+</sup> and use colonies to grow o.n. cultures for plasmid isolation | ||
| + | |||
| + | |||
| + | '''Plates''' | ||
| + | |||
| + | Showed single colony growth on plates with J61002-pME<sup>tal</sup>+RBS-RFP plasmids, and were stored in the fridge for future preculture growth. | ||
| + | |||
| + | :→ The plates with high and low concentration of transformed cells showed colonies in the expected ratio. | ||
| + | |||
| + | :→ On the plates for pZntR<sup>+</sup> and pCueO<sup>+</sup> no colonies were dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed. | ||
| + | |||
| + | :→ The plates with pArsR<sup>+</sup> plasmid showed only a few non-coloured colonies, mostly medium red colour, andn a few dark red colonies. It was already thought that the pArsR is a bit leaky and causes expression of RFP. | ||
| + | |||
| + | '''Over Night Cultures''' | ||
| + | |||
| + | :→ Both cultures of BBa_J61002-J23101 showed growth, and can be used for plasmid isolation. | ||
| + | |||
| + | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | ||
| + | |||
| + | '''Plasmid Purification''' | ||
| + | |||
| + | Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23101 BBa_J23101] constitutive promoter with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit". | ||
| + | |||
| + | * From each tube 4mL of culture was collected in one 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded. | ||
| + | * Plasmids were eluted with 40μL MQ and stored in the fridge | ||
| + | |||
| + | '''New over night cultures (2 days)''' | ||
| + | |||
| + | The six plates containing colonies of E.coli TOP10 with BBa_J61002-pArsR<sup>+</sup>/pZntR<sup>+</sup>/pCueO<sup>+</sup> were used to inoculate four tubes with 5 mL LB-amp<sub>100</sub> for each promoter. | ||
| + | |||
| + | :→ Instead of an over night incubation, the incubation time was increased to two nights, because it was sunday. | ||
| + | |||
| + | '''Buoyancy Test''' | ||
| + | |||
| + | Four tubes with 6mL LB-amp<sub>100</sub> were inoculated with E.coli TOP10 pNL29, pSB1AC3-M/L-GVP, and pSB1AC3-RFP yesterday afternoon. All tubes had cell growth of comparable size, but no OD<sub>600</sub> measurement was made. | ||
| + | |||
| + | :→ The tubes were placed in a foam support to let the cells come to rest and placed at room temperature (a picture was taken as time zero, see below). | ||
===Transporters=== | ===Transporters=== | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
| + | *'''Transform ''E. coli'' with pSB1AC3-fMT, MymT, SmtA ligations''' | ||
| + | ** Use chemically competent cells and transform using heat shock (37°C) | ||
| + | **Transform ligation mixtures of | ||
| + | ::pSB1AC3 + MymT | ||
| + | ::pSB1AC3 + SmtA | ||
| + | ::pSB1AC3 + fMT | ||
| + | ::pSB1AC3 (self ligation control) | ||
| + | ::pSB1AC3-H + RFP (31-07-09; positive control) | ||
| + | ::MQ (negative control) | ||
| + | **Plate on LBAmp and put o/n in 37°C | ||
===Vectors=== | ===Vectors=== | ||
Latest revision as of 22:41, 22 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
- → DONE Isolate plasmids from E.coli Top10 BBa_J23101 cells
- → DONE Plate E.coli Top10 BBa_J23101 cells from o.n. culture
- → TODO
Pellet remaining cells for short storage in -20 box(not needed)
- → DONE Place the four test cultures with and without GVP on table at room temperature (make foto)
- → DONE Look at plates stored at 37C with BBa_J61002-pArsR+/pZntR+/pCueO+ and use colonies to grow o.n. cultures for plasmid isolation
Plates
Showed single colony growth on plates with J61002-pMEtal+RBS-RFP plasmids, and were stored in the fridge for future preculture growth.
- → The plates with high and low concentration of transformed cells showed colonies in the expected ratio.
- → On the plates for pZntR+ and pCueO+ no colonies were dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed.
- → The plates with pArsR+ plasmid showed only a few non-coloured colonies, mostly medium red colour, andn a few dark red colonies. It was already thought that the pArsR is a bit leaky and causes expression of RFP.
Over Night Cultures
- → Both cultures of BBa_J61002-J23101 showed growth, and can be used for plasmid isolation.
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23101 BBa_J23101] constitutive promoter with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
- From each tube 4mL of culture was collected in one 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Plasmids were eluted with 40μL MQ and stored in the fridge
New over night cultures (2 days)
The six plates containing colonies of E.coli TOP10 with BBa_J61002-pArsR+/pZntR+/pCueO+ were used to inoculate four tubes with 5 mL LB-amp100 for each promoter.
- → Instead of an over night incubation, the incubation time was increased to two nights, because it was sunday.
Buoyancy Test
Four tubes with 6mL LB-amp100 were inoculated with E.coli TOP10 pNL29, pSB1AC3-M/L-GVP, and pSB1AC3-RFP yesterday afternoon. All tubes had cell growth of comparable size, but no OD600 measurement was made.
- → The tubes were placed in a foam support to let the cells come to rest and placed at room temperature (a picture was taken as time zero, see below).
Transporters
Metal Accumulation
- Transform E. coli with pSB1AC3-fMT, MymT, SmtA ligations
- Use chemically competent cells and transform using heat shock (37°C)
- Transform ligation mixtures of
- pSB1AC3 + MymT
- pSB1AC3 + SmtA
- pSB1AC3 + fMT
- pSB1AC3 (self ligation control)
- pSB1AC3-H + RFP (31-07-09; positive control)
- MQ (negative control)
- Plate on LBAmp and put o/n in 37°C
Vectors
Dry
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