Digestion scheme:

Incubate samples for 60 min at 37 °C, then induce a heat shock for 2 min at 45 °C. 5 µL marker and 10 µL of the samples are loaded onto the SDS-gel.

Digestion scheme:

Dilution of primers:

Stock: dilution of primers with aqua bidest. to 100 µM
Working stock: 1:10 (10 µL primer stock, 90 µL water)

Dilution of templates:

• Bacillus gDNA: 1:50 → 10 µL gDNA + 490 µL water
• pKH-STP: 10 ng/µL
• PSG1164: 232 ng/µL → 1:20 → 10µL plasmid + 190 µL water

Reaction Mix

Purification according to the "Purification of PCR Products (QIAquick Gel Extraction Kit) short protocol".

Samples:

• 1' Cat-Resistence
• 2' amyE-Gene
• 3' GFP
• 4' (=2+4+6)-Pool, all fragments

Salmonsperm for 10 min on 95 °C, then on ice
*pMA12 - cut with HindIII + EcoRI
**pMA12 - cut by Daniel Schindler

• resuspend yeast pellet in all samples
• induce heat shock at 42 °C for 35 min
• centrifuge for 30 sec - 1 min at 13.000 g
• resuspend pellet in 200 µL sterile water
• plate on selective medium
• incubate at 30 °C for 3 days

Miniprep of Yeast Culture 2'', 3'' and 4''

• flush cells from plate with 2ml water
• centrifuge for 1min at 11.000 rpm
• pellet cracking: add glass balls
• → miniprep accoding to the miniprep procotol (Omega)

Test PCR with Miniprep 2'', 3'' and 4''

Dilution of elution to 20ng/µl:

• 2'': 72ng/µl*X=10*20ng/µl → 5,3:4,7
• 3'': 38ng/µl*X=10*20ng/µl → 3:7
• 4'': 66ng/µl*X=10*20ng/µl → 2,7:3,7

PCR for 3h

Amplification of plasmid in E.Coli DH5Alpha.

• transformation of 2'', 3'' and 4'' in DH5Alpha
• plating on each ampicilin and canamycin plates

Inoculation of preculture plates: 6ml LB + 6µl Ampicilin + colony clone

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

DNA-concentration: 660ng/µl

Dilution of Primers

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

Reaction Mix

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

Dilution of primers

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

PCR Hag-Flanks Mix

Results

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

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

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

Salmonsperm for 10min on 95°C, then on ice - all attempts on ice!
*pMA12 - cut with HindIII + EcoRI
**pMA12 - cut by Daniel Schindler

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

From 10.04.2014, performed by Daniel

Plasmids prepped above have been transformed into E.coli, performed by Daniel
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

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.

The three obtained fragments together with the NcoI-restricted 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.

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= 7.5ng/µl
Transformation of E.coli DH5a with 5 µl DNA (37.5 ng)
Over night incubation on LB-Amp plates.

The transformation of the E.coli cells was not successful. So it has been repeated with the rest of the prepped 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 2kb fragments

The PCR-product has been checked on a agarose gel (3 µl + 1 µl 6x Loading-Dye) Expected band: 2000bp 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.

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 restricted fragment was purified with the Gel Extraction Kit (c = 24 ng/µl).

(20ng 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 restricted 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 DH5a with 10 µl of the ligation-mix. The transformation was incubated on a LB-Amp-plate at 37°C over night.

The plates from 15.12 were empty which indicates an unsuccessful ligation. A new ligation was performed after calculation with a Ligation calculator: UT Dallas

The ligation was incubated at roomtemperature for 1.5 h. Afterwards it was stored at -20°C.

The plasmids were prepped and restricted with ask Daniel for restriction used enzymes! Although there is a linear fragment it seems too small. The expected size of the plasmid should be 6666 bp.

The plasmids prepped on Saturday seem to be smaller as expected. For this reason a new attempt to eliminate the restriction sites was carried out. The amplification of fragment 2 (with primers iGEM 003 and iGEM 010 on 15.04.2014) did not happen without any problems such as unspecific smaller bands in addition to the expected one (ca 1000 bp). So this time primer iGEM 003 was replaced by iGEM 002 which would result in a smaller fragment of approximately 600 bp. Later on the fragments 1 and 3 together with the new created one, the restricted pMa12 construct and the amyE-fragment (to overlap the region between fragments) would be transformed into S. cerevisiae for recombination.

PCR was performed according to the PCR protocol and the PCR-products were analyzed on a 1% agarose gel.

The transformed yeast cells were incubated at 30°C.

Some new competent E. coli DH5a cells were made because the last batch of cells have not yielded in good results in the last few transformations. The cells were made according to the protocol in the method section below. To test the cells they were plated on normal LB, LB-Amp, LB-Kan and a test-trafo was carried out with pMa12 which provides an ampicillin resistance.

The transformation of E. coli DH5a with the ligation of the pMAD-vector and the hag-flank-construct was not successful. Therefore the PCR for amplification of the construct was repeated by using the hag / flank-parts or the construct itself as template.

PCR was performed according to the "PCR Q5 elongation for 2kb fragments" protocol and the PCR-products were analyzed on a 1% agarose gel.

Since the last ligation attempts were not successful the plasmid pMAD is restricted by us instead using the restricted pMAD from Florian. The PCR-fragments of the hag-flank-construct were purified (PCR1: c = 62 ng/µl, PCR2: c = 55 ng/µl, PCR3: c = 46 ng/µl) and restricted with the enzymes BamHI and NcoI.

The restriction was incubated at 37°C for 1 h and stored at -20°C.