Team:Groningen/Notebook/4 August 2009

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Revision as of 14:40, 4 August 2009

Wet

GVP Cluster

All precultures containing a plasmid with a promoter have grown to the expected density! Time for some plasmid isolation to be carried out...

→ DONE weekly meeting (9 to 12)

→ DONE isolate plasmids from precultures

→ DONE perform a restriction on the medium promoter containing plasmids with SpeI and PstI

→ DONE purify cut vectors with miniprep (11bp fragment should be lost)

→ TODO perform a ligation between cut vector and GVP fragment

→ TODO transform E.coli TOP10 competent cells with ligation product

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:pSB3K3 pSB3K3] and [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] with high, medium and low constitutive promoters 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 3mL of culture was collected in a 1.5mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
• Plasmids were eluted with 50μL MQ and stored in the fridge

Concentration

The concentration of isolated plasmid was determined with the use of a nano-drop.

pSB3K3-highP in MQ

• 11.3 ng/μL
• 0.227 (260/280)
• 0.114 (260/230)

pSB3K3-mediumP in MQ

• 17.2 ng/μL
• 0.344 (260/280)
• 0.201 (260/230)

pSB3K3-lowP in MQ

• 12.9 ng/μL
• 0.257 (260/280)
• 0.143 (260/230)

pSB1AC3-highP in MQ

• 114.0 ng/μL
• 2.279 (260/280)
• 1.335 (260/230)

pSB1AC3-mediumP in MQ

• 55.9 ng/μL
• 1.118 (260/280)
• 0.614 (260/230)

pSB1AC3-lowP in MQ

• 47.9 ng/μL
• 0.957 (260/280)
• 0.512 (260/230)

Restriction for Assembly

The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016].

• 16μL plasmid in MQ (0.8-1.6μg)
• 2μL Fast digest buffer
• 1μL PstI fast digest enzyme
• 1μL SpeI fast digest enzyme

Vector Purification

Cut vector purification was performed on the restricted plasmid [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoters with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit". The 11bp fragment between the SpeI and PstI restriction sites should be lost in this way, and gel purification may be omitted.

• After adding 200μL of resuspention buffer, 200μL lysis buffer and 350μL neutralization buffer the solution was centrifuged for 10min. and the standard protocol was followed.
• Cut vectors were eluted with 20μL MQ and stored in the fridge

Concentration

The concentration of purified cut vector was determined with the use of a nano-drop.

pSB1AC3-highP-res. in MQ

• 14.9 ng/μL
• 0.278 (260/280)
• 0.172 (260/230)

pSB1AC3-mediumP-res. in MQ

• 19.7 ng/μL
• 0.394 (260/280)
• 0.204 (260/230)

pSB1AC3-lowP-res. in MQ

• 17.5 ng/μL
• 0.349 (260/280)
• 0.210 (260/230)

Over Night Cultures

Over night cultures in 4mL LB-amp₁₀₀ medium were prepared from the remaining overnight cultures of:

→ E.coli TOP10 pSB3K3-high const. promoter (kan.)

→ E.coli TOP10 pSB3K3-med. const. promoter (kan.)

→ E.coli TOP10 pSB3K3-low const. promoter (kan.)

and put in the 37°C waterbath at 200 rpm. This was done because the low copy plasmid resulted in a to low isolated concentration. Longer growth and elution in less MQ should increase the recovered amount.

Over night culture in 5mL LB-amp₁₀₀ medium was prepared from the following glycerol stock:

→ E.coli TOP10 GVP1 (amp.)

and put in the 37°C waterbath at 200 rpm.

Transporters

GlpF

Restriction, ligation and transformation. Done as noted at 31 July .

Metal Accumulation

Fusion ArsR with MBP

Restriction

Restriction

Vectors

Dry