Team:Groningen/Notebook/14 August 2009

GVP Cluster

 * → ✅ make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown)
 * → ✅ isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures
 * → ✅ make a planning for next week

Plates

Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth.


 * → The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at 37°C for an additional 4 hours to be sure. Still no growth after addition incubation at 37°C, can be due to high energy cost of vesicle production resulting in the death of the cells!

Over Night Cultures


 * → All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. From each culture 800uL was added to 300uL 87% glycerol and frozen in liquid nitrogen. Stocks were stored at position 21 to 24 in the -80°C fridge.



Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids BBa_J61002 with high, medium and low constitutive promoters with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".


 * From each tube 8mL of culture was collected in a 2.0mL 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

HmtA
After 2 weeks of PCR-ing plasmid isolations there is a product worth wile to send for sequencing. 4,5,6, and 8

In the first 4 lanes PCR2 (261bp) is created from HmtA-AC3 plasmid with PstI 's left in place. In lane 5 till 8 PCR1 (1153bp)is made from HmtA-AC3 plasmid with PstI 's left in place to make mega primers to mutate the restriction sites.

In the lower left quarter is a PCR on HmtA-AC3 plasmid with PstI 's left in place with VF2 VR and failed. The second lower quarter is a XbaI restriction control on plasmids 4,5,6 and 8 not completely digested but indicating the construct is correct in the way that there is a restriction site of the bio brick, meaning the EcoRI mutation got removed.

Metal Accumulation

 * ligate MymT in pSB1AC3
 * Purify mymT-RBS-pre/suff from gel using Roche Gel Extraction kit
 * Elute in 25uL TE buffer (pH8)
 * Digest mymT-RBS-pre/suff with EcoRI and Spe I
 * Ligate with pSB1AC3/EcoRI-SpeI

Dry
Jasper changed the model to take dimeric binding of ArsR into account and to ignore ArsD, as well as putting an image map on-line as the first step towards a clearer interface. The new model leads to a number of third order equations, which means that it is no longer feasible to give closed-form solutions, but it does seem perfectly feasible to compute the solutions numerically (as demonstrated by the updated calculator on the accumulation page).