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From 2012.igem.org

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You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Contents 1 Labwork 1.1 May and June 2012 1.2 July 2012 1.3 August 2012 1.4 September 2012 2 Methods and Protocols 2.1 Plasmid Preparation 2.1.1 Plasmid Preparation of E.coli (Mini Preparation) 2.1.2 Plasmid Preparation of Saccharomyces cerevisia 2.2 Transformation 2.2.1 Yeast Transformation 2.2.2 E.coli Transformation 2.3 PCR 2.4 Culture Medium 2.5 Agar Plate 2.5.1 LBampicillin-Agar 2.5.2 SCD-Agar 2.5.3 YEPDG418-Agar 2.6 Gel Electrophoresis 2.7 Kit

Labwork

May and June 2012

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

• Purchasing of the equipment (reaction tubes, glass bottles, pipette tips,..)

• Primer design

July 2012

• Plasmid isolation of p426, p423, pUD8e, pUD22e from E.coli

• Isolation of chromosomal DNA of CEN.PK2-1C

• Trials to get the genes, promoters and terminators via PCR

August 2012

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

Templates	Amplified DNA Fragments
synthesized sequence of HMG-CoA	HMG-CoA
synthesized sequence of GGPPS	GGPPS
synthesized sequence of Cps/Ks	CPS/KS
chromosomal DNA of CEN.PK2-1C	ERG20

• Linearization of p426 and p423 with SpeI and XhoI

• 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
    

• 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
    

• 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)
    

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

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

• 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
    

September 2012

• 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

• 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)

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

• 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)

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

• 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
  • amplification of KO

• 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

• Midi-preparation of plasmids for sequencing
  • biobrick plasmids
  • mevalonate overexpression plasmid

• 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

• 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)

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

Transformation

Yeast Transformation

E.coli Transformation

PCR

Culture Medium

Full Medium (YEPD) for Yeast
Yeast Extract	1 % (weight/volume)
Pepton	2 % (w/v)
Glucose	2 % (w/v)

Synthetic Complete Medium (SC) for Yeast
Yeast Nitrogen Base	0.17 % (w/v)
Ammoniumsulfate	0.5 % (w/v)
Glucose	2 % (w/v)
Amino Acid Mix*	50 ml/l
Histidin**	0.25 mM
Tryptophan**	0.19 mM
Leucin**	0.35 mM
Uracil**	0.44 mM

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

SOC-Medium for Regeneration of transformed Escherichia coli`s after Electroporation
Trypton	2 % (w/v)
Yeast Extract	0.5 % (w/v)
NaCl	10 mM
KCl	2,5 mM
MgCl2	10 mM
MgSO4	10 mM
Glucose	20 mM

pH has to be regulated to pH=6.8-7.0

Full Medium (LB) for E.coli
Yeast Extract	0.5 % (w/v)
Trypton	1 % (w/v)
NaCl	0.5 % (w/v)

pH has to be regulated with NaOH to pH=7.5

Every cluture medium has to be autoclaved to be sterile.

Agar Plate

LB_ampicillin-Agar

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

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

Agarose Gel (1x)
TAE puffer	1x
Agarose	1 % (w/v)

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

TAE Puffer (50x) for Gel Electrophoresis
EDTA	18,6 g
Tris	242g
Glacial Acetic Acid	57,2 ml
Purified Water	1000ml

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

Kit

PCR Purification Kit
Gel Extraction Kit
Midi Plasmid Preparation Kit