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Labwork

May and June 2012

1. Arrangements for labwork
   

   • preparation of competent cells (E.coli, S.cerevisiae), agarose plates (LB, YEPD, SCD-ura,…), medium for E.coli and S.cerevisiae

2. Purchasing of the equipment (reaction tubes, glass bottles, pipette tips,..)
3. Primer design

July 2012

1. Plasmid isolation of p426, p423, pUD8e, pUD22e from E.coli
2. Isolation of chromosomal DNA of CEN.PK2-1C
3. Trials to get the genes, promoters and terminators via PCR

August 2012

1. PCR of the genes, promoters and terminators
   

   • all genes (without KO and KAH), promoters and terminators could be amplified

2. Linearization of p426 and p423 with SpeI and XhoI

Gel 1: PCR of the promoters and terminators.
There are shown the DNA fragments of the PCR with appropriate annealing temperatures. All promoters and terminators could be amplified. As templates were used the plasmids pUD8e(tHXT7, pPFK1, tTAL1, pTPI1) and pUD22e (tPFK2, tPDC1, pPGI1).

Gel 2: Linearization of p426, p423, pSB1C3 and PCR of the genes HMG-CoA, GGPPS, ERG20, CPS/KS.
As a control it is also the circular p426 and p423 shown. The linear p426 and p423 are about 6300 bp long. pSB1C3-linear means the restriction of the plasmid with EcoRI and PstI in this case (it is 2050 bp long). As PCR templates for HMG-CoA, GGPPS and CPS/KS were used the synthesized DNA fragments and for ERG20 chromosomal DNA of CEN.PK2-1C.

3. Biobrick production of the genes HMG-CoA, ERG20, CPS/KS
   

   • restriction of 3 µg of the genes with EcoRI and PstI
     
   • ligation of biobrick genes with linear pSB1C3
     
   • transformation of the ligation in E.coli
     
   • plasmid isolation of E.coli clones
     
   • control restriction of biobrick plasmids with EcoRI and SpeI
     

Gel 3: Control restriction of pSB1C3-HMG-CoA, pSB1C3-ERG20 and PCR of GGPPS.
The biobrick plasmids were cut with EcoRI and SpeI. The correct sizes of pSB1C3-HMG- CoA from two clones could be observed (2050 bp + 1605 bp), as well as the correct sizes of pSB1C3-ERG20 (2050 bp + 1080 bp). However the fragment of 1080 bp is very hard to see on this picture. With the PCR of GGPPS could not only amplified the correct fragment (870 bp) but also a fragment of 3000 bp.

Gel 5: Control restriction of pSB1C3-CPS/KS.
The biobrick plasmids were cut with EcoRI and SpeI. The correct sizes of pSB1C3-CPS/ KS from two clones could be observed (2050 bp + 2900 bp). The plasmid of clone 3 is the pSB1C3 without the insert.

4. Formation of the mevalonate overexpression plasmid via gap repair

   • first and second yeast transformation with equimolar quantities of DNA fragments for mevalonate overexpression (p426 with 7 inserts): only very small colonies could grow after the first and second transformation
   • using pure GGPPS (purification of a preparative gel) for the third yeast transformation: normal size of the colonies
   • inoculation of several clones of the third yeast transformation
   • plasmid preparation of the clones
   • transformation of the plasmids in E.coli
     

5. Amplifying pSB1C3 for biobrick production

   • trials to amplify pSB1C3, whose blunt ends were ligated and transformed in E.coli
   • pSB1C3 should be linearized by EcoRI and PstI : did not work (two fragments instead of one)
     

Gel 4: Purified GGPPS and linearization of pSB1C3.
The 870 bp fragment of the GGPPS PCR (see gel 3) were cut out from a preparative gel and then purified with a gel extraction kit (The purified GGPPS could be used for transformation, but not for constructing a biobrick. Therefore another PCR was made with a very short synthesis time. So only the 870 bp fragment was obtained.)
Because of lack of pSB1C3 the remained linear pSB1C3 was ligated and transformed in E.coli. After isolation of the plasmid it was restricted with EcoRI and PstI. But there was not only the linear fragment of 2050 bp but also another fragment about 4000 bp.

1. • preparative gel of the linear fragment: very low concentration of linear pSB1C3 (was not sufficient for ligation)

6. GC analysis

   • GC analysis of the wild type CEN.PK2-1C (standard GGOH): as expected no GGOH could be observed

7. Assembly of the KO and the KAH fragments

   • amplification of the fragments via PCR (there are four fragments of each gene with an overhang to the fragment beside of 30 bp)
   • Gibson assembly of the fragments of KO and KAH: did not work
     

Gel 6: PCR of KO and KAH fragments
Each gene consists of four fragments, which have a 30 bp overhang to the fragments beside. In order to do the Gibson Assembly, the fragments were amplified via PCR.

Gel 7: Gibson Assembly of KO and KAH
The assembly did not work, because there are only the 500 bp of the single fragments.

September 2012

1. Formation of the mevalonate overexpression plasmid via gap repair

   • plasmid isolation (p426 with 7 inserts) from E.coli
   • control restriction of the plasmids with EcoRI and SpeI: one clone out of 10 got the right sizes

Gel 11: Possible mevalonate overexpression plasmids from E.coli.
After yeast transformation with p426 and 7 inserts, isolation of the plasmids, transformation in E.coli and isolation of them, they were loaded on a gel with a negative control (p426) in order to see which plasmid runs slowest. In this case it is the plasmid of clone 10.

Gel 12: Control restriction of possible mevalonate overexpression plasmids.
The plasmids of clone 1, 5 and 10 were cut with EcoRI. The correct sizes of the mevalonate overexpression plasmids are 7000 bp, 2400 bp and 1900 bp. The plasmid of clone 10 seems to be the correct one.

Gel 13: Control restriction of possible mevalonate overexpression plasmids.
The plasmids of clone 2, 3 and 10 were cut with SpeI. The correct sizes of the mevalonate overexpression plasmids are 7800 bp, 1900 bp and 1610 bp. The plasmid of clone 10 seems to be the correct one. To have a positive control p426 was also cut with SpeI (6300 bp).

1. Ergosterol experiment

   • idea: maybe the clones of the first and the second yeast transformation grow better after ergosterol addition (0,02 g/l)): wildtype with and without ergosterol and one of the clones with and without ergosterol (no significant difference in growth could be observed)

2. GGPPS-PCR

   • PCR of GGPPS with shorter synthesis time in order to get only the correct fragment

3. Amplification of pSB1C3 for biobrick production

   • transformation of pSB1C3-RFP in E.coli
   • isolation of the plasmid from E.coli
   • linearization with EcoRI and PstI: it worked (only the correct fragment was observed)

4. PCR of biobrick-promoters and -terminators

   • PCR of biobrick promoters and terminators that were used to build the mevalonate overexpression plasmid

Gel 10: PCR for biobrick-promoters and –terminators.
There are shown the DNA fragments of the PCR with appropriate annealing temperatures. All promoters and terminators could be amplified, except pHXT7. As templates were used the plasmids pUD8e (tHXT7, pPFK1), pUD22e (tPFK2, pPGK1) and p426 (pHXT7, tCYC1).

Gel 14: PCR for biobrick-pHXT7 and –pPGK1.
There are shown the DNA fragments of the PCR with appropriate annealing temperatures. As templates were used the plasmids pUD22e (pPGK1) and p426 (pHXT7).

1. Assembly of the KO and the KAH fragments

   • trials to assembly the KO and the KAH fragments via PCR (the overhangs were used as primer): KO worked, KAH not

Gel 8: Assembly of KO1/2, KO3/4, KAH1/2 and KAH3/4 via PCR.
Because the Gibson Assembly did not work the assembly was done via PCR. Therefore the 30 bp overhangs were used as primer and the appropriate annealing temperature was chosen.
KO1/2: 500 bp + 500 bp = 1000 bp
KO3/4: 500 bp + 240 bp = 740 bp
KAH1/2: 500 bp + 500 bp = 1000 bp
KAH3/4: 500 bp + 260 bp = 760 bp

Gel 9: Assembly of KO and KAH via PCR.
The assembly of KAH did not work. For the PCR of KO were used the assembled KO1/2 and KO3/4 with an appropriate annealing temperature (the 30 bp overhang was used as primer). The KO fragment (1600 bp) was then amplified via a PCR with normal primers.

1. • amplification of KO

2. Biobrick production of the genes GGPPS, KO and the promoters and terminators

   • restriction of 3 µg of the DNA fragments with EcoRI and PstI
   • ligation of the promoters, terminators, KO and the GGPPS with pSB1C3
   • transformation of the ligation in E.coli
   • plasmid isolation of E.coli clones
   • control restriction of biobrick plasmids with EcoRI and SpeI

3. Midi-preparation of plasmids for sequencing

   • biobrick plasmids
   • mevalonate overexpression plasmid

4. GC

   • GC analysis of the wild type CEN.PK2-1C with p426 and the wild type with the mevalonate overexpression plasmid in SCD-ura
   • standard GGOH

5. Formation of the plasmid for steviol synthesis

   • yeast transformation with equimolar quantities of DNA fragments for steviol synthesis (p423 with 7 inserts)
   • problem: could not assemble KAH, assembled KAH1/2 and KAH3/4 were used (KAH1/2 and KAH3/4 only got a 30 bp overhang instead of a 45 bp overhang): gap repair did not work

Methods and Protocols

Plasmid Preparation

Plasmid Preparation of E.coli (Mini Preparation)

1. transfer 1,5 ml of an overnight culture in a reaction tube
   
2. centrifuge at 8000 rpm at RT for 5 min
   
3. resuspend the pellet in 100 µl solution 1
   
4. addition of 200 µl solution 2
   
5. mix till the solution is clear
   
6. incubate at RT for 5 min
   
7. addition of 150 µl cold solution 3, mix till protein clumps are build
   
8. incubate 10 min on ice, centrifuge 15 min with 10000 rpm
   
9. transfer the supernatant in a clean reaction tube
   
10. fill the reaction tube with 96 % EtOH, mix and let it precipitate at -20 °C for 10 min
    
11. centrifuge 10 min at 10000 rpm
    
12. wash the pellet with 70 % EtOH
    
13. dry the pellet and resuspend it with 30 µl water or TE buffer
    

Solution 1

• 50 mM glucose
• 10 mM EDTA
• 25 mM Tris-HCl pH8

Solution 2

• 0,2 M NaOH
• 1 % SDS

Solution 3

• 3 M KaAc pH 5,5

Plasmid Preparation of Saccharomyces cerevisia

1. overnight culture in 5 ml
   
2. centrifuge 2 ml of the cells for 1-2 min
   
3. wash the cells with water
   
4. resuspend the pellet in 400 µl buffer 1 with RNase
   
5. addition of 400 µl buffer 2 and mix carefully
   
6. addition of 2/3 volume of glass beads
   
7. cell destruction: vibrax the cellsin a 2 ml reaction tube for 5 min at 4°C
   
8. transfer 500 µl of the supernatant in a clean reaction tube
   
9. addition of 250 µl buffer 3, mix, incubate it for 10 min on ice
   
10. centrifuge at 10000 rpm, 15 min
    
11. transfer the supernatant in a clean reaction tube, fill with isopropanol and mix
    
12. centrifuge at 13000 rpm, 30 min
    
13. wash the pellet with cold 70 % EtOH and let it dry
    
14. resuspend the DNA in 30 µl water or TE buffer
    

P1

• 50 mM Tris/HCl pH 8
  
• 10 mM EDTA
• 100 µg/ml RNase A

P2

• 0,2 M NaOH
• 1 % SDS

P3

• 3 M KAc

Transformation

Yeast Transformation

1. Inoculate the synthetic complete medium (SC) with the strain
2. Incubate with shaking overnight at 30°C
3. Harvest the cells at a OD600 0,5-0,6 by centrifugation (3000x g, 5 min, RT)
4. Wash the cells with 0,5 vol of sterile water (resuspend by shaking, centrifugate with 3000x g, 5 min, RT)
5. Resuspend the cells in 0,01 vol of sterile water, transfer the suspension to a reaction tube and pellet the cells (3000x g, 5 min, RT)
6. Resuspend the pellet in 0,01 vol of sterile filtrated FCC (frozen competent cell) solution
7. Aliquot 50 µl of the solution into the reaction tubes
8. Store the cells at -80°C for at least one night (up to one year)
9. Mastermix for the FCC transformation mixture:

1. Prepare DNA aliquots: Solute enough DNA (e.g. 100 ng plasmid) in 54 µl of water
2. Unfreeze the cells in a 37°C block for 15-30 sec
3. Centrifuge the solution at 13000x g for 2 minutes
4. Remove the supernatant
5. Add 306 µl of FCC transformation mixture to the cells
6. Add 54 µl of the DNA to the solution and vortex shortly
7. When all the reaction tubes are prepared, vortex the samples well until all pellets are completely resoluted
8. Incubate the samples for 40 minutes at 42°C in a heating block
9. Centrifuge the cells at 13000x g for 30 sec and pour off the supernatant
10. Resuspend the cells in sterile water by vortexing
11. Spread onto the appropriate medium
12. Let the cells grow at 30°C

Yeast transformation and gap repair (example)

° determination of 50 ng for 360 bp
use of a vector-insert relation of 1:1
samples: linear p426 with 7 inserts

positive control (p426)

negative control (linear p426)

plating the samples on SCD-ura and incubation at 30° C

General procedure of a transformation via gap repair

1. Amplification of the required DNA fragment with homologue ends of the plasmid (in our case homologue ends of the gene/promoter/terminator beside the DNA fragment) via PCR and an agarose gel to review the correct size
   
2. Linearization of the plasmid and an agarose gel for review
   
3. Yeast transformation with the PCR products and the linear plasmid (homologue recombination)
   
4. Selection on an agar plate without the metabolite, which is on the plasmid for yeast selection (in our case it was uracil on p426 and histidin on p423)
   
5. Inoculation of clones and isolation of the plasmids from yeast
   
6. Transformation of a plasmid in E.coli and selection on LB medium with ampicillin (the plasmids have an ampicillin resistance)
   
7. Isolation of the plasmids from E.coli
8. Diagnostic restriction of the plasmid in order to find the correct plasmid with all inserts
   
9. Transformation of the correct plasmid in yeast to do further experiments

E.coli Transformation

a) Production of competent cells

1. Inoculate 400 ml LB medium (pre-warmed to 37°C) in a 1 l flask with 100 µl of a fresh over night culture

2. Harvest the cells at a OD600 0,60-0,65 (in a room with 37°C, sterile)

3. Aliquot the solution in 8 x 50 ml falcons (sterile, pre-cooled on ice)

4. Incubate for 30 min on ice

5. Centrifuge for 12,5 min with 4000x g, 4°C

6. Resuspend the pellets in each 10 ml sterile water (4°C)

7. Centrifuge for 10 min with 4000x g, 4°C

8. Repeat steps 6 and 7 two times

9. Resuspend the cells in 10 ml 10% glycerine (+millipor, sterile, 4°C)

10. Centrifuge for 15 min with 4000x g, 4°C

11. Add 800 µl of 10% glycerine (sterile, 4°C) to the cells

12. Aliquot 40 µl of the solution in each reaction tube (pre-cooled) and freeze them in liquid nitrogen

13. Store them up to 6 months at -70°C

b) Transformation (on ice)

1. Add DNA (e.g. 1 µl of extracted yeast plasmids) to the frozen cells

2. Unfreeze the cells on ice

3. Fill the cells into cuvettes for electroporation

4. Electroporation (for 2 mm cuvettes: voltage I = 2,5 kV; resistance R = 2000 ohm; electrical current A = 25 µF; for 1 mm cuvettes: I = 2,2 kV; resistance R = 2000 ohm; electrical current A = 25 µF)

5. Fill the cells with 1 ml of SOC medium back in a reaction tube

6. Shake them for 1 hour at 37°C

7. Centrifuge for 1 min 8000x g

8. Decant the supernatant

9. Spread onto the appropriate medium

PCR

Culture Media

pH has to be regulated with KOH to pH=6.3
° contains no His, Leu, Trp and Uracil
** addition of this components depents on the respective selection medium

pH has to be regulated to pH=6.8-7.0

pH has to be regulated with NaOH to pH=7.5

Every cluture medium has to be autoclaved to be sterile.

Agar Plate

LB_ampicilline-Agar

Add 2 % agar to LB-medium. After autoclaving and cooling-down to 60 °C steril ampicillin is added. Plates were poured.

LB_{chloramphenicol}-Agar

Add 2 % agar to LB-medium. After autoclaving and cooling-down to 60 °C ethanolic chlorampenicol solvation is added.

SCD-Agar

Add 2 % agar to SCD-medium. After autoclaving and cooling-down steril amino acid solution is added. Dependent on the respective selective medium Histidin (0.25 mM), Trypthophan (0.19 mM), Uracil (0.44 mM) or Leucin (0.35 mM) are added. Plates were poured.

YEPD_G418-Agar

Add 2 % agar to YEPD-medium. After autoclaving and cooling-down sterile G418 (final concentration 2g/l) is added. Plates were poured.

Gel Electrophoresis

Solve agarose in TAE by boiling it. After cooling-down to 55-60 °C gel is poured.

pH has to be regulated with glacial acetic acid to pH=8.

Gels were run with a tension of 80-140 V.

Kits

PCR Purification Kit from Qiagen
Gel Extraction Kit from Qiagen
Midi Plasmid Preparation Kit from Qiagen