Team:TU Darmstadt/Labjournal/Metabolism

From 2012.igem.org

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! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
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| Fragment 1 || 40.3
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| Fragment 2 || -
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| Fragment 2 || 62.1
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! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
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| Mutated tphA1 || 86.1
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==week 4 (04.-08.06.12)==
==week 4 (04.-08.06.12)==

Revision as of 13:08, 15 September 2012

Protocols Metabolism


Contents

week 1 (14.-18.05.12)

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
    • Both PCR products were purified via gel extraction
    • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
tphA1 0.6

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
    • Both PCR products were purified via gel extraction
    • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
tphA3 0.1

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
    • Both PCR products were purified via gel extraction
    • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
tphB 0.1

Other

Biobrick Concentration [ng/µl]
BBa_K316003 114.9
BBa_J23100 450.2
BBa_B0015 314.1
BBa_J61101 86.1

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 40.3
Fragment 2 62.1
  • 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 86.1

week 4 (04.-08.06.12)

Other

Plamid backbone Concentration [ng/µl]
pSB1C3 -
Insert Concentration [ng/µl]
xylE-dT -

week 5 (11.-15-06.12)

Other

week 6 (18.-22.06.12)

  • No work progress

week 7 (25.-29.06.12)

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)

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 -

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

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 -

week 11 (23.-27.07.12)

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


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


week 13 (06.-10.08.12)

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 -
Miniprep Concentration [ng/µl]
pSB1C3-tphA2-prefix/suffix -
  • Preparation for sequencing
    • Sequence was confirmed

week 14 (13.-17.08.12)

aroY

Miniprep Concentration [ng/µl]
tphA2 gene synthesis -

Other

Plamid backbone Concentration [ng/µl]
J61002 -

week 15 (20.-24.08.12)

Other

Midiprep Concentration [ng/µl]
pPR-IBA2 -
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 -
Miniprep Concentration [ng/µl]
J61002-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 -
Miniprep Concentration [ng/µl]
J61002-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 -
Miniprep Concentration [ng/µl]
J61002-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]
tphB with RBS -
Miniprep Concentration [ng/µl]
J61002-RBS-tphB -

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 -
Miniprep Concentration [ng/µl]
J61002-RBS-aroY -


Over expression

tphA1

  • PCR on pSB1C3-tphA1
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphA1 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
tphA1_over-ex -
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA1_over-ex -

tphA2

  • PCR on pSB1C3-tphA2
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphA2 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
tphA2_over-ex -
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA2_over-ex -

tphA3

  • PCR on pSB1C3-tphA3
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphA3 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
tphA3_over-ex -
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA2_over-ex -

tphB

  • PCR on pSB1C3-tphB
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphB and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
tphB_over-ex -
Miniprep Concentration [ng/µl]
pPR-IBA2-tphB_over-ex -

aroY

  • PCR on pSB1C3-aroY
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-aroY and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanoprop
PCR product Concentration [ng/µl]
aroY_over-ex -
Miniprep Concentration [ng/µl]
pPR-IBA2-aroY_over-ex -

week 17 (03.-07.09.12)

Operon construction

RBS-tphA1-RBS-tphA2

  • Restriction digest of J61002-RBS-tphA1 by EcoRI and SpeI
  • Purification of insert RBS-tphA1 RBS-tphA1 (cut with EcoRI and SpeI) via gel extraction
Insert Concentration [ng/µl]
RBS-tphA1 (cut with EcoRI and SpeI) -
Miniprep Concentration [ng/µl]
J61002-RBS-tphA1-RBS-tphA2 -

RBS-tphA3-RBS-tphB

  • Restriction digest of J61002-RBS-tphA3 by EcoRI and SpeI
  • Purification of insert RBS-tphA3 (cut with EcoRI and SpeI) via gel extraction
Insert Concentration [ng/µl]
RBS-tphA3 (cut with EcoRI and SpeI) -
Miniprep Concentration [ng/µl]
J61002-RBS-tphA3-RBS-tphB -

RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB

Insert Concentration [ng/µl]
RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI) -
Miniprep Concentration [ng/µl]
J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB -

Over expression and protein purification

tphA2


tphB


SDS-PAGE of tphB overexpression and tphA2 overexpression respectively

  • SDS-Page according to standard protocol
A
Results of the overexpression tphA2/tphB
Band Sample Time [h]
1 tphB 0
2 tphB 1
3 tphB 2
4 tphB 3
5 tphA2 0
6 tphA2 1
7 tphA2 2
8 tphA2 3
9 Protein Marker-

week 18 (10.-17.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 19 (17.-21.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

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