The new control PCR was not successful either.

The PCR with TG_PheA-Arc1p-C-8x FP und TG_PheA-Arc1p-C-8x RP should generate the 2346 bp fragment for Gibson Assembly from the synthesized template.

The used Primer concentration was 1 µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.

The amplified fragment was purified on an agarose gel on which it showed the correct size. It was extracted from the gel for further use in a Gibson Assembly yielding a concentration of 258 ng/µL.

The pET-28a vector was cut with XhoI and NdeI in order to create the ends needed for the integration of the prepared PheA-Arc1p-C-8x construct via Gibson Assembly

The reaction mixture was incubated for 3 h at 37 °C and 350 rpm. The resulting linearized vector was purified using an agarose gel from which it was extracted for further use yielding a concentration of 24 ng/µL.

The reaction mixture was incubated for 1 h at 50 °C and 350 rpm. E. coli Top10 electrocompetent cells were transformed with a 1:3 and a 1:6 dilution of the mixture, incubated for 1 h at 37 °C and plated out on LB-Kan⁵⁰ plates that were incubated overnight at 37 °C.

Miniprep according to the miniprep kit protocol (Omega) with overnight preculture.

DNA-concentration: 660 ng/µL

Dilution of Primers

• dilution with aqua bidest to 100 µM
• dilution 1:10 → 10 µL primers (100 µM) with 90 µL aqua bidest

Reaction Mix

Incubation for 60 min at 37 °C. Then a heat shock was induced for 2 min at 45 °C.

Six clones of the transformed Top10 cells were picked and used to inoculate overnight cultures (6 x 5 mL).

Dilution of primers

• Hag I: 601,7 ng/µL
• Hag II: 548,2 ng/µL
• Flank I: 226 ng/µL
• Flank II: 221 ng/µL

PCR Hag-Flanks Mix

Results

Expected bands: 2 kb band (whole Flank-Hag fragment)

The gel shows the successful amplification of the Hag-flank construct.

Gel extraction of 2,2 kb band:
27,6 ng/µL

Attempts with 2 positive clones (2.1 NcoI cut & 2.3 NcoI cut)

Salmonsperm for 10 min on 95 °C, then on ice - all attempts on ice!
*pMA12 - digestion with HindIII + EcoRI
**pMA12 - parallel digestion of HindIII and EcoRI

• Resuspend yeast pellet in attempts
• Heat shock at 42 °C for 35 min
• Centrifugation for 30 sec - 1 min at 13.000 g
• Resuspend pellet in 200 µL sterile water
• Plate yeast on selective medium
• Incubate at 30 °C till monday

The plasmids were purified from the overnight cultures using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.

40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-Arc1p-C-8x-pET28a from two different clones, incubated for 1 h at 37 °C and plated out on LB-Kan⁵⁰ plates that were incubated overnight at 37 °C.

The extracted 2000 bp fragment was used as a PCR template for amplification of the Hag-flank construct for further cloning steps.

The plasmid isolation was carried out according to the miniprep kit protocol.

Plasmids prepared above have been transformed into E.coli.
Colonies on transformation plates have been inoculated in shaking culture (16 colonies).

Miniprep according to the miniprep kit protocol (Omega).
2.1 and 2.3 different positive colonies from 09.04.2014.

Overnight digestion of two samples with the highest DNA-concentration (2.1 2 and 3.1 A1

5 mL of LB-Kan⁵⁰ medium were inoculated with a clone from 14.6 bearing the PheA-Arc1p-C-8x-pET28a plasmid.

As template the restriction of the sample 3.1 A1 has been used. The mutagenesis primers have been chosen together with fitting reverse primers to create fragments without the NcoI-restriction sites. These fragments should further be transformed into yeast and assembled into the whole plasmid without the restriction sites for NcoI.

Every PCR has been carried out in double and was further analyzed on a 1% gel.

Using the preculture from 14.7 60 mL of LB-Kan⁵⁰ medium were inoculated with 600 µL of the preculture and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The culture was cooled down to 18 °C and the protein production was induced with 12 µL of 500 mM IPTG. Over night the culture was incubated at 18 °C and 220 rpm. A glycerolstock of the strain containing the PheA-Arc1p-C-8x-pET28a plasmid was stored at -80 °C.

The three obtained fragments together with the NcoI-digested plasmid pMA12 have been transformed into S. cerevisiae to get the whole plasmid again. The whole procedure has been done according to the protocol.

The transformed yeast cells are incubated at RT over the easter holidays.

The cell culture from 14.8 was centrifuged (17000 rpm, 20 min, 4 °C) and the pellet resuspended in 3 mL buffer A. Lysozyme was added to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 °C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.

60 mL LB-Kan⁵⁰ medium was inoculated from the glycerolstock prepared in 14.8 and incubated at 37 °C and 220 rpm over night.

10 x 500 mL LB-Kan⁵⁰ medium were inoculated with 5 mL of the overnight culture prepared in 14.9 and incubated at 37 °C and 220 rpm until OD₆₀₀ reached 0.5. The flasks were cooled to 18 °C and protein production was induced by adding 100 µL 500 mM IPTG. The cultures were incubated over night at 18 °C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 °C), resuspended in buffer A and stored at -20 °C until further use.

Miniprep of Transformed Yeast S.cerevisiae from 21.04.2014 and Trafo of E.coli DH5Alpha.
Miniprep was performed according to the miniprep protocol (Omega).

Plasmid pMa12 with 3 inserts (cat, amyE and gfp) with removed NcoI restriction sites.
Concentration after plasmid prep= 7.5 ng/µL
Transformation of E.coli DH5α with 5 µL DNA (37.5 ng)
Over night incubation on LB-Amp plates.

The resuspended cells from 14.10 were lysed using a french press. The lysate was incubated on ice with DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C). The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-8x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.

The transformation of the E.coli cells was not successful. So it has been repeated with the rest of the prepared plasmid.

The concentration of the Hag-Flank-construct was too low (c = 5.8 ng/µL) to use it for the ligation into pMAD. A PCR has been run to increase the amount of the construct.

PCR Q5 elongation for 2 kb fragments

The PCR-product has been checked on an agarose gel (3 µL + 1 µL 6x Loading-Dye) Expected band: 2000 bp is there, but also 2 additional bands A band with the expected size of ca 2000 bp is visible but there are also two additional DNA-fragments with a lower size. The rest of the PCR-product (47 µL) has been separated on a gel and the expected DNA-fragment has been excised and purified with the Gel Extraction Kit. The purification led to a concentration of 45 ng/µL.

The used Primer concentration was 1 µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.

The amplified fragment was purified on an agarose gel on which it showed the correct size. The extracted linear template was then digested to degrade the original unmutated plasmid using DpnI.

The reaction mixture was incubated at 37 °C and 300 rpm for 1 h and afterwards heated to 80 °C for 20 min

In order to create the circular plasmid 100 ng of the linear template were mixed with 1 µL 10x T4-Ligasebuffer, 1 µL T4-Ligase and water to give a total volume of 10 µL. This mixture was incubated for 9 h at 16 °C. The resulting plasmid was dialysed with water to decrease the salt concentration for the following transformation of E. coli Top10 electrocompetent cells with the plasmid.

Seven small colonies grew on the plate. The plate was further incubated until the afternoon. Each 6 mL LB-Amp have been inoculated with one of the colonies and incubated over night at 37°C.

The hag-flank-construct which was amplified yesterday has been digested with NcoI and BamHI to create sticky ends for the following ligation.

The restriction was carried out at 37°C for 45 min and the digested fragment was purified with the Gel Extraction Kit (c = 24 ng/µL).

(20 ng of vector x kb size of insert/kb size of vector) x molar of ratio of (insert/vector)

0.6 µL of the pMAD-vector (30 ng) and 1 µL of the digested hag-flank-construct (18 ng) were used for the ligation.

The ligation-mix was incubated at roomtemperature for 1 h followed by a transformation of E. coli DH5α with 10 µL of the ligation-mix. The transformation was incubated on a LB-Amp-plate at 37 °C over night.

Six clones of the transformed Top10 cells from 14.12 were picked and used to inoculate overnight cultures (6 x 5 mL).