Sponsors

Strain:

E. coli strain:

• MC1061 : F− araD139, Δ(ara-leu)7696,galE15, galK16, Δ(lac)X74, rpsL (Strr), hsdR2 (rK−mK+), mcrA mcrB1
• DG1(Delphi genetics): mcrA Δ (mrr-hsdRMS-mcrBC, modification-,
  restriction-) F80lacZDM15 Δ lacX74 recA1 araD139 Δ (ara-leu)7697 galU galK rpsL endA1 nupG
• TOP10 (Invitrogen) : F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-
• MG1655 : F- λ- ilvG- rfb-50 rph-1
• MG1655 ΔtldD::frt-cm-frt

Saccharomyces cerevisae strain:

• 23344C :ura3

Plasmid:

pCP20:

pCP20 has the Flp recombinase gene repressed by CI857ts which is not functional at a temperature higher or equal to 42°C

pFL44S :

pFL44S is a shuttle plasmid between yeast and bacteria. It has the ampicillin resistance gene and the bacterial origin of replication colE1. For selection in yeast it has the ura3 gene. It has also the 2micron yeast origin of replication.

pKD46:

pKD46 expresses the Red system under control of the well-regulated promoter pBAD (induced by arabinose and repressed by glucose). The replication origin of the plasmid is also thermo sensitive. It is not replicated at a temperature higher or equal to 42°C.

pSB1A3:

pSB1A3 is one of the standard iGEM plasmid which has a resistance gene to ampicillin. It is composed of four unique restriction sites:

• EcoRI (prefix)
• XbaI (prefix)
• SpeI (suffix)
• PstI (suffix)

PCR Topo® XL plasmid (Invitrogen):

This plasmid is used in the Topo® XL PCR Cloning Kit. For the cloning the plasmid is linearized and topoisomerase l is activated. The vector includes the following features:

• ccdB gene for positive selection
• Kanamycin and Zeocin™ resistance genes
• EcoRI sites flanking the PCR product insertion site for easy excision of inserts
• M13 forward and reverse primer sites for sequencing

Primers:

The following primers were produced by Sigma-Aldrich (option desalt)

Yeast cloning:

• PKD46-FOR:

5’-gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggctgccacctgcatcgatttat-3’

• PKD46-REV:

5’-cgtgagttttcgttccactgagcgtcagaccccgtagaaagagttttcgttccactgagc-3’

• PFL-FOR:

5’-gcctttttacggttcctggc-3’

• PFL-REV:

5’-tttctacgggggtctgacgc-3’

• PCP-FOR:

5’-tggctcttgtatctatcagtgaagcatcaagactaacaaatcagccaaacgtctcttcag-3’

• PCP-REV:

5’-ggggctgtatgcacaaagcatcttctgttgagttaagaacttatatgcgtctatttatgtagg-3’

In green is the 40 homologous nucleotides need for the yeast cloning.

Yeast cloning verification:

• FLP/CI-FOR:

5’-acatggcgagttttgacgag-3’

• FLP/CI-REV:

5’-accacactagagaacatactg-3’

pINDEL sequencing:

• pID-seq1:

5’-aggatcttcacctagatcctt-3’

• pID-seq2:

 5’-gatgggctagtcaatgataatta-3’

• pID-seq3:

5’-ccgttacgtaggtaggaatc-3’

• pID-seq4:

5’-agatggggatggggcagtc-3’

• pID-seq5:

5’-gatttcggatcaacgttcttaat-3’

• pID-seq6:

5’-caatcactttcgtctactcc-3’

• pID-seq7:

 5’-ccagatatttcgccgcgac-3’

• pID-seq8:

5’-cggggccagcaaaaaatcca-3’

• pID-seq9:

5’-ccctgatttttcaccacccc-3’

• pID-seq10:

5’-cttccgaaaatgcaacgcga-3’

Biobrick FRT'-CM-FRT':

• FRT’-CM-FRT’-FOR:

5’-gaagttcctatactttttagagaataggaacttcgttgatcgggcacgtaagagg-3’

• FRT'-CM-FRT'-REV :

5’-gaagttcctattctctaaaaagtataggaacttcttattacgccccgccctgcc-3’

In green is the frt’ sequence which is not homologous with the chloramphenicol gene.

• TOPO-FRT'-CM-FRT'-FOR:

5'-tccggcaaaaaagggcaaggtgtcaccaccctgcccttttcgccagtgtgctggatttc-3'

• TOPO-FRT'-CM-FRT'-REV:

5’-tgcagcggccgctactagtactctagaagcggccgcgaatgatggatatctgcagatttc-3’

In red is the iGEM restriction sites (prefix and suffix) and in green is the mutation made to suppress the EcoRI restriction site (in the Topo® XL PCR plasmid).

Biobrick FRT'-CM-FRT' sequencing (primer M13 as described in Topo® XL PCR Kit):

• FRT’-CM-FRT’-SEQ-FOR:

5’-gaggaaacagctatga-3’

• FRT’-CM-FRT’-SEQ-REV:

 5’-gaccggcagcaaatg-3’

Media:

Bacteria, Luria-Bertani (LB):

• 10g/l tryptone;
• 5g/l yeast extract;
• 5g/l NaCl
• (+12g/l of Agar for solid medium)

Yeast:

Rich medium:

• 10g/l of Yeast Extract
• 10g/l of bactopeptone
• 20g/l of glucose

Minimal medium:

• 20g/l of glucose
• 0.67 g/l Difco Yeast Nitrogen Base w/o Amino acid
• 15g/l of agar

Solution:

• Mg(SO4)2 (10-2M)
• TSS composed of (for 10ml): 8,5ml of LB medium; 500µl of dimethylsulfoxide; 500µl of MgCl2 (1M); 1g polyethylene glycol 8000
• Isopropyl-ß-D-galactoside (IPTG) (1000X): 1M
• Dimethylsulfoxyde (Sigma Aldrich D2650)

Antibiotics (stock):

• Ampiciline 100mg/ml
• Kanamycine 50 mg/ml
• Chloremphenicol 20 mg/ml
• Tetracycline 15mg/ml

The antibiotics were diluted 1000X in appropriate medium.

Enzymes:

• EcoRI, XbaI, SpeI, PstI, BstXI 10U/µl (Roche)
• rAPID Alkaline Phosphatase 1U/µl (Roche)
• Taq polymerase Gotaq 5U/µl (Promega)
• Taq polymerase Phusion 2U/µl (Finnzymes)
• T4 DNA ligase 1U/µl  (Roche)

PCR:

Finnzymes, Physion® Hot Start High-Fidelity DNA polymerase (Ref: f-540S):

Reaction solution:

• 10µl of buffer5X
• 1µl of dNTPs (10mM each)
• 0.25µl of primer 1 (20µM)
• 0.25µl of primer 2 (20µM)
• 1µl of DNA
• 0.5µl of Taq polymerase Phusion
• 1.5µl of DMSO (optional)
• 37 (or 35.5 if DMSO) µl of H20

Program:

• 1:98°C 30sec
• 2:98°C 10sec
• 3:58°C 30Sec
• 4:72°C X min, 30sec/kb (30 cycles from step2)
• 5:72°C 10min

Promega, GoTaq® Hot Start Polymerase (ref: M5001):

Reaction solution:

• 10µl of buffer10X (green or greenless)
• 1µl of dNTPs (10mM each)
• 3µl of MgCl2
• 0.5µl of primer 1 (20µM)
• 0.5µl of primer 2 (20µM)
• 1µl of DNA
• 0.25µl of Taq polymerase Phusion
• 1.5µl of DMSO (optional)
• 33.75 (or 32.25 if DMSO) µl of H20

Program:

• 1:94°C 5min
• 2:94°C 30sec
• 3:60°C 30sec
• 4:72°C X min, 1min/kb (30 cycles from step2)
• 5:72°C 10min

Miniprep kit:

 Zymo Research, ZyppyTM Plasmid Miniprep Kit (ref: D4036)

Midi prep kit:

Sigma-Adrich, GenEluteTm HP Plasmid Midiprep Kit (ref:Na0200-1KT)

Gel purification kit:

 Sigma-Aldrich, GenEluteTm PCR Clean-up (ref:NA1020)

Yeast transformation by lithium acetate : Gietz version. (culture for 100ml)

Cells preparation:

• From one fresh dish make a preculture of your strain on rich medium (10ml) and incubate it overnight at 30°C and preincubate 100ml of rich medium at 30°C.
• In the morning dilute your preculture in the rich medium and incubate it at 30°C during three hours in a way your OD600nm is towards 0.5-0.7.
• Centrifuge your culture at 3500g for 5 minutes and remove supernatant.
• Resuspend pellet in 1ml steril H20, transfer in a 2ml eppendorf, centrifuge 10 seconds, remove supernatant, resuspend in 1ml water, recentrifuge 10 seconds and remove water.
• Resuspend pellet in 1ml AcLi/TE, centrifuge 10 seconds, remove supernatant and resuspend the pellet in 0.5ml AcLi/TE.

DNA transformation:

• At 50mL of suspension add in order:
• 5mL sonicated DNA at 10mg/ml, mix delicately.
• ±1mg DNA to transform, mix delicately.
• 300ml PEG/AcLi/TE 40% solution, homogenize.
• Incubate 30 minutes at 30°C.
• Incubate 15 minutes at 42°C
• Centrifuge 10 seconds, empty the tube, add 1ml water and directly empty the tube. Then resuspend the cells in 100µl of water.
• Spread on yeast minimal plates and incubate at 30°C for 3 days.

Used Solutions:

• 10x AcLi: 1M AcLi pH 7.5.
• 10x TE: 0.1M Tris pH 7.5: 0.01M EDTA pH 7.5.
• PEG 4000 50%.
• AcLi/TE: 1/10 vol 10X AcLi + 1/10 vol 10x TE in steril water.
• PEG 40%/AcLi/TE: 8/10 vol PEG 4000 50% + 1/10 vol 10x AcLi + 1/10 vol 10x TE.