Team:Wageningen UR/Notebook/Proj1/May

May 3

Meeting with modeling expert Hans Stigter from Biometris.

May 17

Biobrick parts were resuspended from the biobrick source plate in 10 µL dH2O. 1.5 µL of this suspension was used to transform chemically competent E. coli TOP10 cells. The bricks were added to 50 µL of cells in duplo. The mixtures were incubated on ice for 30 min. Then a heat shock was carried out at 42°C for 1 min. 50 and 250 µL of the mixtures were plated out on LB + amp and grown o/n at 37°C.

May 20

50 µL of competent E. coli TOP10 cells were transformed with 1.5 µL solutions of the BioBrick parts in duplo, by incubation on ice for 15-40 min. followed by a heat shock at 42°C during 1 min. The following parts had to be inserted: BBa_I0460, -F2621, -F1610, -K325219, -K325210, -PO412 and -K325909. 250 µL SOC medium was added and they were incubated for 1.5 hour at 37°C (300 rpm). 50 µL of these inoculated plates A, 250 µL of these inoculated plates B.

May 23

Of the Top10 BBa_F2621 and BBa_K325219 transformants, which were made on May 20, a few colonies were found. More colonies were found of the BBa_K325210 and BBa_PO412 transformants. The rest of the transformations has not yielded growth.

May 24

Following the protocol of May 20, E. coli were transformed with BBa_F2621 (marker: Amp), -325219 (marker: Chl), -325210 (marker: Chl), -F1610 (marker: Kan), -K325909 (marker: Chl) and -I0460 (marker: Kan).

By colony PCR some of colonies were screened. The Invitrogen protocol was used. The PCR thermocycler, of Biorad, was set to the following values for 30 cycles: 94°C at the denaturation, 50°C at the annealing and 72°C at the elongation step. Of the following transformants 4 colonies were taken: BBa_F2621, -32519, -32510, -E0422 and -I0462.

PCR cycling:

1. hotstart 94°C for 3 min. 2. denaturing 94°C for 30 45s 3. annealing 55°C for 30 s 4. elongation 72°C for 1m30s 5. pause 4°C

The 4 colonies BBa_E0422 transformants and 3 colonies of the BBa_I0462 transformants were confirmed. For the rest of the samples the electrophoresis gel was not clear.

Liquid cultures of the BBa_E0422 and BBa_I0462 transformants were made. Tubes with 10 mL of LB medium and 10 µL Amp (stock) were inoculated with the corresponding colonies and untransformed Top10 cells - as a negative control. These were incubated overnight.

May 25

The liquid cultures were made successfully on May 24 - unlike the other incubations, the negative control showed no growth.

With colony PCR the cultures were screened. The Biorad PCR thermocycler was set up in the following way: 30 cycles of 94°C at the denaturation, 50°C at the annealing and 72°C at the elongation step (duration of it was 2.5 min. because the length the luciferase gene is around 2600 bp).

May 30

PCRs were perfomed on BBa_I0462 transformants in liquid culture and colonies, a BBa_I0462 plasmid and a control.

Two tubes with 10 mL of LB medium containing Amp were inoculated with BBa_10462 transformant colonies. These were incubated at 37°C on a shaker overnight.

May 31

Of both overnight grown cultures 5 mL was taken to miniprep with the Fermentas GeneJET Miniprep kit.

Out of the miniprep came a 100 ng/µL and a 130 ng/µL plasmid solution, as determined with the Nanodrop spectrophotometer.

The plasmids, 10 µL of the solution, of both samples were digested with the restriction enzymes EcoRI and PstI separately and together at 37°C for two hours.

The success of the PCRs of May 30 and the digestions was found out by having run an agarose gel at 100 V during one hour. Except for the PCR that was performed with seq-primers (the seq-primers probably did not work), the PCRs and digestion went well.

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