17th July

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9:45 inoculated 30 ml LB amp in 37ºC shaking incubator
pSB1AK3
pSB1AC3
pSB1AT3

11:30 purified plasmids
(in conctrary to protocol i accidentally added >350µl lysis buffer, so i also added 450µl neutralisation buffer) Yield of 100 µl elution:
BBa_B0032: 21.2 ng/µl
BBa_K145015: 33.0 ng/µl
BBa_K145201: 27.8 ng/µl

BBa_K145280 30ml culture induced with 1mM IPTG at 14:40

At 17:45 I was done with the plasmid purification of the strains inoculated this morning and the GFP-LVA + TetR generator strain inoculated yesterday. The DNA concentrations of the 95µl volumes are listed below: pSB1AK3: 72,2 ng/µl; 260/280=2,18;260/230=2,22;
pSB1AC3: 79,2 ng/µl; 260/280=2,19;260/230=2,18;
pSB1AT3: 76,4 ng/µl; 260/280=2,14;260/230=2,10;
BBa_K145280: 141,9 ng/µl; 260/280=1,79;260/230=1,67;
Note that the backbone plasmids have a significantly lower concentration than the GFP-LVA generator strain. This is partly because the GFP-LVA strain had a higher cell density (longer incubation). But also the lysate was much clearer than the other strains. This is also reflected in the 260 nm ratios, which are much lower (=better) for the K145280 strain. The 30 ml cultures were divided into 15 ml fractions and centrifuged. Elution was done with 50 µl and these fractions were then combined to get the (approx.) 95µl end product. This yield will allow approximately 14 assemblies per backbone.

Calin

Riboregulator scheme 1 and scheme 2 Matlab scripts finished. Ordered lambda red plasmids, (pKD46, pKD3, pKD4) from [http://cgsc2.biology.yale.edu Yale CGSC]. Helped Sriram with transformations. Used DH5alpha comptetent cells to transform:
BBa_I714031 - OriT-R
BBa_J23100 - strong promoter
BBa_E0840 - GFP generator
BBa_I13522 - pTet GFP