Team:Ruperto Carola/Notebook
Weekly reports
Lab setup
The practical phase of our project was heralded by the great ceremony where 4 team members were chosen to embark upon the journey to the university’s lab supply store. They came back rewarded richly with all the goods a scientist can dream of. Petri dishes, pipet tips, syringes, cannulas, gloves, tubes, reaction vessels, inoculation loops, ethanol —a whole lot of ethanol, gas cartridges, filters, well plates, pipettes in all sizes, parafilm, cuvettes and extraction kits. The inauguration was finished by filling the shelves and cabinets and by distributing the benches.
Preparation of chemically competent DH5αTM E.coli cells (Thermo Fischer Scientific)
Prepared LB, SOC media and LB plates with different antibiotic markers. Liquid Media Left under foaming hood at Room temperature after autoclaving, plates in fridge or cooling room at 4°CBacterial cells were cultured in 2 ml LB over-night at 37°C shaking at 180 RPM.1 ml of the over-night culture were then diluted up to 200 ml of LB medium, theculture was subsequently grown up to an O.D600=0.6 (about 2.5-3 hours). Subsequently the cultured bacteria were pelleted and re-suspended in 15 ml TSS buffer (1XLB, 5% DMSO, 10 mM MgCl2, 10 mM MgSO4, 10 mM PEG 4000). The TBS (TSS) bacterial suspension were left on oce to cool down for 10 minutes. The suspension was divided into individual 100 μl aliquots that were kept ondry ice and subsequently preserved at -80°C.
2 Batches were prepared in parallel.
Batches were both tested next day by transforming 3 aliquots each with BBa_J04450. Alliquots were thawed on ice and after gently mixing by pipetting up and down split into 2*50 μl (for negative control). 1 or 10 pg DNA were added and mixed by gently flicking tubes. Protocol for Transformation of bacterial cells with non-ampicillin-antibiotic marker was followed.
Red colonies were counted to get following average efficiencies:
Batch 1: \(7,1*10^6 \frac{cfu}{μg}\)
Batch 2: \(1,2 *10^7 \frac{cfu}{μg}\)
Preparation of 416
The plasmid has oris for E. coli as well as for S. cerevisiae. The yeast ori is centromeric which causes the plasmid to be single. Furthermore, it has an uracil marker for selection. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
Preparation of 415-GDP
The plasmid has oris for E. coli as well as for S. cerevisiae. The yeast ori is centromeric which causes the plasmid to be single copy. Furthermore, it has an leucine marker for selection and a strong constitutive glyceraldehyde-3-phospate dehydrogenase promoter in front of the MCI. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
Preparation of 426-GDP
The plasmid has oris for E. coli as well as for S. cerevisiae. The yeast ori is a high copy configuration. Furthermore, it has an uracil marker for selection and a strong constitutive glyceraldehyde-3-phospate dehydrogenase promoter in front of the MCI. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
Preparation of 425-GDP
The plasmid has oris for E. coli as well as for S. cerevisiae. The yeast ori is a high copy configuration centromeric. Furthermore, it has an leucine marker for selection and a strong constitutive glyceraldehyde-3-phospate dehydrogenase promoter in front of the MCI. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
Preparation of pYM-N9
The plasmid has oris for E. coli as well as for S. cerevisiae. It has an leucine marker for selection and a strong constitutive glyceraldehyde-3-phospate dehydrogenase promoter in front of the MCI. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
Preparation of 316-Fus1-lacZMT
The plasmid has oris for E. coli as well as for S. cerevisiae. The yeast ori is centromeric which causes the plasmid(CEN6) to be single copy. Furthermore, it has an uracil marker for selection and a strong constitutive glyceraldehyde-3-phospate dehydrogenase promoter in front of the MCI. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
qMaM57
This plasmid contains a mCherry tag followed by an ADH1 terminator and a kanamycin resistance all flanked by standardised S2/S3 primer sites. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
pMaM375
This plasmid contains a GFP tag followed by an ADH1 terminator flanked by standardised S2/S3 primer sites. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
pKS133
This plasmid contains a hygromycine resistance gene flanked by standardised S2/S3 primer sites. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
pKS134
This plasmid contains a clonNAT resistance gene flanked by standardised S2/S3 primer sites. To get proper amounts of the plasmid Promega Midipreps were performed. A 100 ml overnight culture was pelleted at 5000 g. The pellet was resuspended in 3 ml of resuspension solution and added to 3 ml of cell lysis solution and slowly inverted four times. After 3 minutes neutralization solution was added and inverted 7 times. The lysed cells were centrifuged at 15000 g. The supernatant was filled onto a binding column. After washing the column the plasmids were eluted with 20 μl water and measured with a nano photometer.
Yeast culture growth
Yeast growth in delta Leu medium with 5-FOA was measured in 96 well plates with concentrations of 1 g per litre, 0,9 g per litre, 0,8 g per litre, 0,7 g per litre, 0,6 g per litre, 0,5 g per litre. Measurement was running over 18 hours.
Ste2 knock-out
pKS133 was used as template for a Q5 PCR with S2/S3 primers with overhangs for chromosome 9 Ste2 flanking regions to generate the Ste2 knock out cassette. Less than 1 μg of template, 10 μl Hi-Fi 5x buffer, 2 μl MgCl2(50 mM), 2,5 μl of each primer (10 μM),5 μl dNTPs (10 mM), 5 μl (5 M) of betain, 21,75 μl H2O and 0,25 μl 2 u/μl of Q5 polymerase und keine Eier. The cycle program was set up to 97 °C for 2 min, the following 3 cycles were repeated 35 times 97 °C for 30 sec, 68 °C 30 sec, 72 °C for 40 sec, and finally 72 °C for 5 min and kept cool at 4 °C. A 4 μl sample of the product was loaded on a 2 % agarose gel to prove the PCR has worked. The product was cleaned up with the Enzo PCR &Gel Clean-up Kit. The PCR product was diluted 1 to 2 in binding buffer and loaded onto the clean up column. After washing the column the product was eluted and its concentration was measured with the nanophotometer. Yeast was made competent with PEG 4000, lithium acetate and a
Yeast culture growth
Yeast synthetic dropout medium was obtained by taking yeast nitrogen base concentrate and diluting it from 4 x to 1 x with delta Leucine Tryptophan concentrate or delta Leucine Tryptophan Uracil concentrate and glucoses concentrate. Two strains F102+FPD and F102+FPD A75 were cultured in YPD(full medium), delta Leu Trp, delta Leu Trp Ura and delta Leu Trp with 1 g 5-FOA per litre in 10 ml/50 ml cultures each. Cultures were grown and optical density over 2 days.
Yeast culture growth
Yeast synthetic dropout medium was obtained by taking yeast nitrogen base concentrate and diluting it from 4 x to 1 x with delta Leucine Tryptophan concentrate or delta Leucine Tryptophan Uracil concentrate and glucoses concentrate. 1 ml of F102+FPD and F102+FPD A75 were inoculated in 100 ml delta Leu Trp Ura, delta Leu Trp and 30 ml full medium. Cultures were measured optical density over 2 days.
Yeast culture growth
Yeast synthetic dropout medium was obtained by taking yeast nitrogen base concentrate and diluting it from 4 x to 1 x with delta Leucine Tryptophan concentrate or delta Leucine Tryptophan Uracil concentrate and glucoses concentrate. 1 ml of F102+FPD and F102+FPD A75 culture were inoculated in 100 ml of delta Leu Ura, delta Leu and full medium with concentrations 1 g per litre, 0.5 g per litre, 0.25 g per litre, 0.125 g per litre, 0.0625 g per litre and 0.03125 g per litre. I am working here now for over 40 h. I don't know for how long I can go on. I want to see the sun just once again. Cultures were measured optical density over 2 days.
Yeast plating
Plating of F102+FPD and F102+FPD A75 in delta Leu Ura plates for F102+FPD and F102+FPD A75 followed imaging of delta Leu Ura. Growth measuring of F102+FPD and F102+FPD A75 delta Leu and delta Leu Ura over two days. Delta Leu plate preparation with 6.7 g per litre YNB, 1.4 g per litre delta YSD, 20 g per liter glucose, 20 g per litre agar and 100 mg per litre extra histidine. 1 ml of F102+FPD and F102+FPD A75 over night culture were grown and measured optical density in 100 ml delta Leu Ura, delta Leu and full medium over three days.
Yeast culture growth
100 ml YPD medium were inoculated with 1 ml F102+FPD and F102+FPD A75 cultures. 50 ml full YSD medium inoculated with 50 μl F102+FPD and F102+FPD A75 cultures. 50 ml delta Leu Ura medium inoculated with F102+FPD and F102+FPD A75 cultures. Optical density was measured over 3 days.
Yeast culture growth
50 μl F102+FPD were inoculated in 3 ml of delta Leu, delta Leu Ura, YPAD and full medium and optical density was measured over 3 days. 50 μl F102+FPD were inoculated in 3 ml of delta Leu, delta Leu Ura, YPAD and full medium and optical density was measured over three days. 100 μl of each preparation were plated on petri dishes with the specific medium with same auxotrophy as in liquid selection medium after one day.
Yeast culture growth
50 μl F102+FPD were inoculated in 3 ml of delta Leu, delta Leu Ura, YPAD and full medium and optical density was measured over one day. 100 μl of each preparation plus delta Leu of days before were plated on petri dishes with the specific medium with same auxotrophy as in liquid selection medium one day later. After two more days (three after beginning) the in delta Leu and YPD grown strains were tested in 1 g per litre 5-FOA.
Ste2 amplification
Ste2 was amplified via PCR. Less than 2 μg of template, 20 μl Hi-Fi 5x buffer, 5 μl of each primer (10 μM), 10 μl dNTPs (10 mM), 10 μl (5 M) of betain, 43,5 μl H2O and 0,5 μl 2 u/μl of Q5 polymerase.
Ste2 amplification
Ste2 was amplified via PCR out of the FDP plasmid. Less than 2 μg of template, 30 μl Hi-Fi 5x buffer, 7,5 μl of each primer (10 μM), 1,5 μl dNTPs (10 mM), 100 μl H2O and 1,3 μl 2 u/μl of Q5 polymerase. Ste2 was amplified via PCR. Less than 2 μg of template, 30 μl Hi-Fi 5x buffer, 7,5 μl of each primer (10 μM), 1,5 μl dNTPs (10 mM), 100 μl H2O and 1,3 μl 2 u/μl of Q5 polymerase. Both products were cut with EcoRI since the products are almost same sized but have unequal cutting sites.
Ste2 amplification
Ste2 amplification was repeated due to failure of the previous one. Ste2 was amplified via PCR out of the FDP plasmid. Less than 2 μg of template, 30 μl Hi-Fi 5x buffer, 7,5 μl of each primer (10 μM), 1,5 μl dNTPs (10 mM), 100 μl H2O and 1,3 μl 2 u/μl of Q5 polymerase. Ste2 was amplified via PCR. Less than 2 μg of template, 30 μl Hi-Fi 5x buffer, 7,5 μl of each primer (10 μM), 1,5 μl dNTPs (10 mM), 100 μl H2O and 1,3 μl 2 u/μl of Q5 polymerase. Both products were cut with EcoRI since the products are almost same sized but have unequal cutting sites.
CPEG
Circular Polymerase Extension Cloning (CPEG) of Ste2 in the FDP integration cassette. 0,5 μl of Ste2, 5 μl primer, 25 μl 2 x Q5 master mix and 19,5 μl H2O 0,5 μl of FDP, 5 μl primer, 25 μl 2 x Q5 master mix and 19,5 μl H2O
Yeast culture growth
50 μl F102+FPD were inoculated in 3 ml of delta Leu, delta Leu Ura, YPAD and full medium and optical density was measured over one day. 100 μl of each preparation plus delta Leu of 09.18 were plated on petri dishes with the specific medium with same auxotrophy as in liquid selection medium one day later. After two more days (three after beginning) the in delta Leu and YPD grown strains were tested in 1 g per litre 5-FOA.
CPEG again
Circular Polymerase Extension Cloning (CPEG) of Ste2 in the FDP integration cassette again due to failure of in the experiment the day before. 0,5 μl of Ste2, 5 μl primer, 25 μl 2 x Q5 master mix and 19,5 μl H2O 0,5 μl of FDP, 5 μl primer, 25 μl 2 x Q5 master mix and 19,5 μl H2O