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From 2012.igem.org

Contents 1 week 1 (14.-18.05.12) 2 week 2 (21.-25.05.12) 3 week 3 (28.05.-01.06.12) 4 week 4 (04.-08.06.12) 5 week 5 (11.-15-06.12) 6 week 6 (18.-22.06.12) 7 week 7 (25.-29.06.12) 8 week 8 (02.-06.07.12) 9 week 9 (09.-13.07.12) 10 week 10 (16.-20.07.12) 11 week 11 (23.-27.07.12) 12 week 12 (30.07.-03.08.12) 13 week 13 (06.-10.08.12) 14 week 14 (13.-17.08.12) 15 week 15 (20.-24.08.12) 16 week 16 (27.-31.08.12) 16.1 Operon construction 17 week 17 (03.-07.09.12) 18 week 18 (10.-17.09.12) 19 week 19 (17.-21.09.12)

week 1 (14.-18.05.12)

tphA1

• No work progress

tphA2

• No work progress

tphA3

• No work progress

tphB

• No work progress

aroY

• No work progress

Other

• Reconstitution of C. testosteroni KF-1 according to DSMZ [http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/engl_Opening.pdf protocol]
• Cultivation of C. testosteroni KF-1 on agar plates with Medium 1
• Production of chemically competent E. coli DH5α and E. coli BL21(DE3)pLysS cells

week 2 (21.-25.05.12)

tphA1

• Isolation of the gene from C. testosteroni KF-1 genome using colony-PCR
  • Annealing temperature: 49 °C
  • Primer: tphA1-l-F and tphA1-l-R

tphA2

• No work progress

tphA3

• Isolation of the gene from C. testosteroni KF-1 genome using colony-PCR
  • Annealing temperature: 59 °C
  • Primer: tphA3-l-F and tphA3-l-R

tphB

• Isolation of the gene from C. testosteroni KF-1 genome using colony-PCR
  • Annealing temperature: 59 °C
  • Primer: tphB-l-F and tphB-l-R

aroY

• No work progress

Other

• Transformation and midi prep of all used Biobricks
  • Concentrations measured by Nanoprop

Biobrick	Concentration [ng/µl]
BBa_K316003	-
BBa_J23100	-
BBa_B0015	-
BBa_J61101	-

week 3 (28.05.-01.06.12)

tphA1

• Two PCRs were performed to mutate the PstI site in the wild type gene. The primer tphA1-l-PstI(99)-R and tphA1-l-PstI(99)-F respectively introduced a BsaI site
  • tphA1 fragment 1
  • PCR on tphA1 isolated from C. tesosteroni
    • Annealing temperature: 69 °C
    • Primer: tphA1-l-PstI(99)-R and tphA1-l-R
  • tphA1 fragment 2
  • PCR on tphA1 isolated from C. tesosteroni
    • Annealing temperature: 69 °C
    • Primer: tphA1-l-PstI(99)-F and tphA1-l-F
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
Fragment 1	-
Fragment 2	-

• Both fragments were cut with BsaI in a restriction digest
  • The ligation mix differed from our standard protocol in the following manner
    • 100 ng of fragment 1
    • 200 ng of fragment 2
    • 2 µL of 10x reaction buffer
    • 1 µL of T4 DNA ligase
    • add DI water up to 20 µL
    • incubate for 15 minutes at 37 °C
  • PCR on ligation mix
    • Annealing temperature: 59 °C
    • Primer: tphA1-l-R and tphA1-l-F
  • The PCR product was purified via gel extraction
  • Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
Mutated tphA1	-

tphA2

• No work progress

tphA3

• No work progress

tphB

• No work progress

aroY

• No work progress

Other

week 4 (04.-08.06.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

• Restriction digest of BBa_K316003 by EcoRI and PstI
  • Purification of plasmid backbone pSB1C3 via gel extraction
  • Concentrations measured by Nanoprop

Plamid backbone	Concentration [ng/µl]
pSB1C3	-

• Restriction digest of BBa_K316003 by XbaI and PstI
  • Purification of insert xylE-dT via gel extraction
  • Concentrations measured by Nanoprop

Insert	Concentration [ng/µl]
xylE-dT	-

week 5 (11.-15-06.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

• Restriction digest of BBa_J23100 by SpeI and PstI
• Dephosphorylation of the restriction digest
• Ligation of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
  • Transformation with 2 µL of the ligation mix
• Colony-PCR of the transformation for verification
• After overnight incubation of colony x in LB medium with ampicilin a glycerine stock was made

week 6 (18.-22.06.12)

• No work progress

week 7 (25.-29.06.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

• Funktional testing of BBa_J23100-xylE-dT
  • We inoculated 50 mL LB-medium-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
  • After incubation we centrifuged the culture at 4600x g for 10 minutes
  • We resuspended the pellet with the 1000 µL pipette in 3 mL PBS buffer and added PBS to 120 ml
  • We added 2 mL of 0.5 M catechol solution to the cell suspension
  • We observed a colour change colourless to light yellow

week 8 (02.-06.07.12)

• No work progress

week 9 (09.-13.07.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

• Designing primers with prefix and suffix respectively
• Designing genes (aroY and tphA2 respectivley) according to the biobrick standard for gene synthesis. Gene synthesis was performed by [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/gene-synthesis.html?s_kwcid=TC|17953|geneart||S|b|12191353721 GeneArt®]

week 10 (16.-20.07.12)

tphA1

• PCR on mutated tphA1
  • Annealing temperature: 59 °C
  • Primer: tphA1-Suffix_R and tphA1-l-Prefix
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
Mutated tphA1-prefix/suffix	-

• Restriction digest of mutated tphA1-prefix/suffix with EcoRI and PstI
• Ligation of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was negative

tphA2

tphA3

• PCR on tphA3 isolated from C. testosteroni
  • Annealing temperature: 59 °C
  • Primer: tphA3-Prefix_F and tphA3-Suffix_R
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
tphA3-prefix/suffix	-

• Restriction digest of mutated tphA3-prefix/suffix with EcoRI and PstI
• Ligation of mutated tphA3-prefix/suffix (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-tphA3-prefix/suffix	-

• Restriction digest of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
• Preparation for sequencing
  • Sequence was confirmed

tphB

• PCR on tphB isolated from C. testosteroni
  • Annealing temperature: 59 °C
  • Primer: tphB-Prefix and tphB-Suffix_R
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
tphB_prefix/suffix	-

• Restriction digest of mutated tphB-prefix/suffix with EcoRI and PstI
• Ligation of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was negative

aroY

Other

week 11 (23.-27.07.12)

tphA1

tphA2

tphA3

tphB

• PCR on tphB isolated from C. testosteroni
  • Annealing temperature: 59 °C
  • Primer: tphB-Prefix and tphB-Suffix_R
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
tphB_prefix/suffix	-

• Restriction digest of mutated tphB-prefix/suffix with EcoRI and PstI
• Ligation of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-tphB-prefix/suffix	-

• Restriction digest of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
• Preparation for sequencing
  • Sequence was confirmed

aroY

Other

week 12 (30.07.-03.08.12)

tphA1

• PCR on mutated tphA1
  • Annealing temperature: 59 °C
  • Primer: tphA1-Suffix_R and tphA1-l-Prefix
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
Mutated tphA1-prefix/suffix	-

• Restriction digest of mutated tphA1-prefix/suffix with EcoRI and PstI
• Ligation of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-tphA1	-

• Restriction digest of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
• Preparation for sequencing
  • Sequence was confirmed

tphA2

tphA3

tphB

aroY

Other

week 13 (06.-10.08.12)

tphA1

tphA2

• Reconstitution of the tphA2 gene synthesis
• Transformation of the tphA2 gene synthesis
• Inoculation of 10 mL LB-medium-kanamycin with one colony of the transformation and incubation
• Miniprep of the culture

Miniprep	Concentration [ng/µl]
tphA2 gene synthesis	-

• Restriktion digest of the tphA2 gene synthesis with EcoRI and PstI
• Ligation of the tphA2 gene synthesis and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony XX and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-tphA2-prefix/suffix	-

• Preparation for sequencing
  • Sequence was confirmed

tphA3

tphB

aroY

Other

week 14 (13.-17.08.12)

tphA1

tphA2

tphA3

tphB

aroY

• Reconstitution of the aroY gene synthesis
• Transformation of the aroY gene synthesis
• Inoculation of 10 mL LB-medium-kanamycin with one colony of the transformation and incubation
• Miniprep of the culture

Miniprep	Concentration [ng/µl]
tphA2 gene synthesis	-

• Restriktion digest of the aroY gene synthesis with EcoRI and PstI
• Ligation of the aroY gene synthesis and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was negative

Other

• Restriction digest of BBa_J23100 by EcoRI and PstI
  • Purification of plasmid backbone J61002 via gel extraction
  • Concentrations measured by Nanoprop

Plamid backbone	Concentration [ng/µl]
J61002	-

week 15 (20.-24.08.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

• Designing primers for over expression and operon construction
• Transformation of pPR-IBA2
• Inoculation of 10 mL of LB-medium-chloramphenicol with one colony and incubation
• Midiprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Midiprep	Concentration [ng/µl]
pPR-IBA2	-

• Restriction digest of pPR-IBA2 with EcoRI and PstI
  • Purification of plasmid backbone pPR-IBA2 via gel extraction
  • Concentrations measured by Nanoprop

Plamid backbone	Concentration [ng/µl]
pPR-IBA2	-

week 16 (27.-31.08.12)

Operon construction

tphA1

• PCR on pSB1C3-tphA1
  • Annealing temperature: 59 °C
  • Primer: RBS-tphA1 and Suffix
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
tphA1 with RBS	-

• Restriction digest of RBS-tphA1 with EcoRI and PstI
• Ligation of RBS-tphA1 (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony XXX and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-RBS-tphA1	-

tphA2

• PCR on pSB1C3-tphA2
  • Annealing temperature: 59 °C
  • Primer: RBS-tphA2 and Suffix
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
tphA2 with RBS	-

• Restriction digest of RBS-tphA2 with EcoRI and PstI
• Ligation of RBS-tphA2 (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony XXX and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-RBS-tphA2	-

tphA3

• PCR on pSB1C3-tphA3
  • Annealing temperature: 59 °C
  • Primer: RBS-tphA3 and Suffix
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
tphA3 with RBS	-

• Restriction digest of RBS-tphA3 with EcoRI and PstI
• Ligation of RBS-tphA3 (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony XXX and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-RBS-tphA3	-

tphB

• PCR on pSB1C3-tphB
  • Annealing temperature: 59 °C
  • Primer: RBS-tphB and Suffix
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
tphA1 with RBS	-

• Restriction digest of RBS-tphB with EcoRI and PstI
• Ligation of RBS-tphB (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony XXX and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-RBS-tphAB	-

aroY

• PCR on pSB1C3-aroY
  • Annealing temperature: 59 °C
  • Primer: RBS-aroY and Suffix
• Both PCR products were purified via gel extraction
• Concentrations measured by Nanoprop

PCR product	Concentration [ng/µl]
aroY with RBS	-

• Restriction digest of RBS-aroY with EcoRI and PstI
• Ligation of RBS-aroY (cut with EcoRI and PstI) and pSB1C3 (cut with EcoRI and PstI)
• Transformation of ligation mix
• Colony-PCR for verification of the transformation
  • The PCR was positive
• Inoculation of 10 mL of LB-medium-chloramphenicol with colony XXX and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanoprop

Miniprep	Concentration [ng/µl]
pSB1C3-RBS-aroY	-

Other

week 17 (03.-07.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 18 (10.-17.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 19 (17.-21.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other