Team:Groningen/Notebook/4 August 2009

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...


 * → ✅ weekly meeting (9 to 12)
 * → ✅ isolate plasmids from precultures
 * → ✅ perform a restriction on the medium promoter containing plasmids with SpeI and PstI
 * → ✅ purify cut vectors with miniprep (11bp fragment should be lost)
 * → perform a ligation between cut vector and GVP fragment
 * → transform E.coli TOP10 competent cells with ligation product



Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB3K3 and pSB1AC3 with high, medium and low constitutive promoters with the "Sygma-Aldrich™ 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 pSB1AC3 containing the high, medium and low constitutive promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick 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 pSB1AC3 containing the high, medium and low constitutive promoters with the "Sygma-Aldrich™ 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 . only the inactivation step was not done using a agarose gel but by using a PCR clean up kit (Sigma-Aldrich).

Metal Accumulation
Fusion ArsR with MBP

Restriction


 * incubated 37&deg; 30 min
 * inactivated enzyme at 65&deg; for 5 min

Ligation
 * incubated at 4&deg; overnight

Dry
Jasper did some "maintenance" on the Wiki and created a diagram of the processes involved in arsenic filtering.

Kb made the final changes to the brochure. This is the final version

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