Team:TU Darmstadt/Labjournal/Metabolism

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Labjournal Metabolism

This lab journal describes a isolation and characterisation of the terephthalic acid 1,2-dioxigenase system and an dihydrodiol decarboxylase from Comamonas testosteroni KF-1 in Escherichia Coli. The strain was purchased from DMSZ-German Collection of Microorganism and Cell Cultures (DMSZ no.14576). For more informations you will see our project discription.

Klonierung sascha.png

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 [http://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1.pdf 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 Nanodrop
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 Nanodrop
PCR product Concentration [ng/µl]
tphA3 0.1
A
tphA1 (left band at 1000 bp) and tphA3 (right band at 500 bp) isolated with colony PCR from C. testosteroni KF-1 genome (far left: 1kb DNA ladder, NEB)

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 Nanodrop
PCR product Concentration [ng/µl]
tphB 0.1
A
tphB (both single bands at 1000 bp) isolated with colony PCR from C. testosteroni KF-1 genome (far left: 1kb DNA ladder, NEB)


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. testosteroni
      • Annealing temperature: 69 °C
      • Primer: tphA1-l-PstI(99)-R and tphA1-l-R
    • tphA1 fragment 2
    • PCR on tphA1 isolated from C. testosteroni
      • 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 Nanodrop
PCR product Concentration [ng/µl]
Fragment 1 40.3
Fragment 2 62.1
A
tphA1 Fragment 1 (left) and tphA1 Fragment 2 (right) after PCR (GeneRuler 100bp Plus DNA Ladder)


  • Both fragments were cut with BsaI in a restriction
    • 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 Nanodrop
PCR product Concentration [ng/µl]
Mutated tphA1 86.1

week 4 (04.-08.06.12)

Other

Plamid backbone Concentration [ng/µl]
pSB1C3 42.6
A
restriction of BBa_K316003 using EcoRI and PstI (1kb DNA ladder, NEB)
Insert Concentration [ng/µl]
xylE-dT 22.2

week 5 (11.-15-06.12)

Other

week 6 (18.-22.06.12)

  • No work progress

week 7 (25.-29.06.12)

Other

  • Functional testing of BBa_J23100-xylE-dT
    • We inoculated 2 x 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 each pellet in 3 mL PBS buffer and added PBS to 120 ml
    • We added 2 mL of 0.5 M catechol solution to the first cell suspension
    • We added 2 ml of 0.5 M protocatechuic acid to the second cell suspenion
    • We observed in either colony a colour change from colourless to light yellow.
      • Conclusion: XylE accepted protocatechuic acid as a substrate.
  • Kinetic assay of XylE with protocatechuic acid as a substrate
    • 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 each pellet in 3 mL PBS buffer and added PBS to 15 ml
    • After cell disruption we centrifuged the suspension at 9600 rpm for 20 min and decanted the supernatant in a fresh tube.
    • For the kinetic measurements we prepared the following standard concentrations:
Nummer Standard [mM]
1 1
2 2
3 5
4 10
5 12,5
6 15
7 20
  • For the kinetic assay we measured the absorption at 380 nm with an UV/Vis-spectrometer for 2 min and calculated the initial speed. The gained data was used to created a Michaelis Menten plot.

Michaelis Menten plot of XylE with protocatechuic acid as substrate

[Protocatecuatuic acid] mM Velocity units per minute
1 0
2 0
5 0
10 0
12,5 0,285

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 Nanodrop
PCR product Concentration [ng/µl]
Mutated tphA1-prefix/suffix 62.0

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 Nanodrop
PCR product Concentration [ng/µl]
tphA3-prefix/suffix 30.5
Miniprep Concentration [ng/µl]
pSB1C3-tphA3-prefix/suffix 79.6
  • restriction 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 Nanodrop
PCR product Concentration [ng/µl]
tphB_prefix/suffix 20.3

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 Nanodrop
PCR product Concentration [ng/µl]
tphB_prefix/suffix 52.5
Miniprep Concentration [ng/µl]
pSB1C3-tphB-prefix/suffix 35.8
  • restriction of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
  • Preparation for sequencing
    • Sequence was confirmed

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 Nanodrop
PCR product Concentration [ng/µl]
Mutated tphA1-prefix/suffix 34.2
A
Colony PCR of pSB1C3-tphA1; from left to right: Colony 1-11 (BenchTop 1kb DNA ladder between colony 6 and 7 and on the far right)
Miniprep Concentration [ng/µl]
pSB1C3-tphA1 60.5
  • restriction of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
A
Test restriction digest of psB1C3-tphA1 with EcoRI and PstI (GeneRuler 100bp Plus DNA Ladder, Fermentas)
  • Preparation for sequencing
    • Sequence was confirmed

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 112.6
A
Colony PCR on psB1C3-tphA2. From left to right: colony 1-12 (far right: BenchTop 1kb DNA ladder, Promega)
Miniprep Concentration [ng/µl]
pSB1C3-tphA2-prefix/suffix 111.1
  • Preparation for sequencing
    • Sequence was confirmed

week 14 (13.-17.08.12)

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]
aroY gene synthesis 63.25

Other

Plamid backbone Concentration [ng/µl]
J61002 42.5

week 15 (20.-24.08.12)

Other

Midiprep Concentration [ng/µl]
pPR-IBA2 127
Plamid backbone Concentration [ng/µl]
pPR-IBA2 35.6

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 Nanodrop
PCR product Concentration [ng/µl]
tphA1 with RBS 33.5
Miniprep Concentration [ng/µl]
J61002-RBS-tphA1 79,6

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 Nanodrop
PCR product Concentration [ng/µl]
tphA2 with RBS 46.8
Miniprep Concentration [ng/µl]
J61002-RBS-tphA2 80.3
A
Colony PCR on J61002-RBS-tphA1 and J61002-RBS-tphA2 (GeneRuler 100bp Plus DNA Ladder, Fermentas)

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 Nanodrop
PCR product Concentration [ng/µl]
tphA3 with RBS 26.5
Miniprep Concentration [ng/µl]
J61002-RBS-tphA3 67.5

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 Nanodrop
PCR product Concentration [ng/µl]
tphB with RBS 49.2
Miniprep Concentration [ng/µl]
J61002-RBS-tphB 65.8

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 Nanodrop
PCR product Concentration [ng/µl]
aroY with RBS 55.2
Miniprep Concentration [ng/µl]
J61002-RBS-aroY 77.2
A
Colony PCR on J61002-RBS-tphA3, J61002-RBS-tphB and J61002-RBS-aroY (GeneRuler 100bp Plus DNA Ladder, Fermentas)

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 Nanodrop
PCR product Concentration [ng/µl]
tphA1_over-ex 116.2
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA1_over-ex 98.5

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 Nanodrop
PCR product Concentration [ng/µl]
tphA2_over-ex 63.9
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA2_over-ex 85.2

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 Nanodrop
PCR product Concentration [ng/µl]
tphA3_over-ex 90.4
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA3_over-ex 85.9

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 Nanodrop
PCR product Concentration [ng/µl]
tphB_over-ex 87.5
Miniprep Concentration [ng/µl]
pPR-IBA2-tphB_over-ex 85.2

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 Nanodrop
PCR product Concentration [ng/µl]
aroY_over-ex 105.1
Miniprep Concentration [ng/µl]
pPR-IBA2-aroY_over-ex 92.1

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) 50.2
  • restriction of J61002-RBS-tphA2 EcoRI and XbaI
  • Dephosphorylation of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)
  • Ligation of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)and RBS-tphA1 (cut with EcoRI and SpeI)
  • Transformation of the ligation mix
  • colony-PCR of the transformation for verification
    • The PCR was positive
A
Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-4 (far right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)
Miniprep Concentration [ng/µl]
J61002-RBS-tphA1-RBS-tphA2 112.5

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) 178.9
  • restriction of J61002-RBS-tphB EcoRI and XbaI
  • Dephosphorylation of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)
  • Ligation of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)and RBS-tphA3 (cut with EcoRI and SpeI)
  • Transformation of the ligation mix
  • colony-PCR of the transformation for verification
    • The PCR was positive
A
Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-9 (far left and right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)
Miniprep Concentration [ng/µl]
J61002-RBS-tphA3-RBS-tphB 225.5

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

  • restriction of J61002-RBS-tphA1-RBS-tphA2 by EcoRI and SpeI
  • Purification of insert RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI) via gel extraction
Insert Concentration [ng/µl]
RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI) 129.5
A
restriction of of J61002-RBS-tphA1-RBS-tphA2 by EcoRI and SpeI (Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)
  • restriction of J61002-RBS-tphA3-RBS-tphB EcoRI and XbaI
  • Dephosphorylation of the plasmid backbone J61002-RBS-tphA3-RBS-tphB (cut with EcoRI and XbaI)
  • Ligation of the plasmid backbone J61002-RBS-tphA3-RBS-tphB EcoRI (cut with EcoRI and XbaI)and RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI)
  • Transformation of the ligation mix
  • colony-PCR of the transformation for verification
    • The PCR was positive
A
Colony PCR on J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB; Colony 1-7 from left to right (Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)
Miniprep Concentration [ng/µl]
J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB 312.2

Overexpression

tphA2

  • Inoculate 10 mL of LB-medium-ampicillin with pPR-IBA2-tphA1_over-ex and incubate
  • Over expression according to standard protocol

tphB

  • Inoculate 10 mL of LB-medium-ampicillin with pPR-IBA2-tphA1_over-ex and incubate
  • Over expression according to standard protocol

aroY

  • Inoculate 10 mL of LB-medium-ampicillin with pPR-IBA2-tphA1_over-ex and incubate
  • Over expression according to standard protocol

tphA1

  • Inoculate 10 mL of LB-medium-ampicillin with pPR-IBA2-tphA1_over-ex and incubate
  • Over expression according to standard protocol

tphA3

  • Inoculate 10 mL of LB-medium-ampicillin with pPR-IBA2-tphA1_over-ex and incubate
  • Over expression according to standard protocol

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 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]-

SDS-PAGE of overexpression from all five genes

  • SDS-Page according to standard protocol
A
Results of the overexpression aroY/tphA3/tphA1/tphA2/tphB/
Band Sample Time [h]
1 aroY 0
2 aroY 3
3 tphB 0
4 tphB 3
5 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker] -
6 tphA1 0
7 tphA1 3
8 tphA2 0
9 tphA2 3
10 tphA3 0
11 tphA3 3
12 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]-

week 18 (10.-17.09.12)

Purification of aroY

A
Fractions of the aroY purfication
Band Sample Fraction
1 aroY 1
2 aroY 2
3 aroY 3 and 4 together
4 aroY 5 and 6 together
5 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]-

Purification of TphA3

A
Fractions of the TphA3 purfication
Band Sample Fraction
1 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]
2 TphA3 Cell suspension
3 TphA3 Cytoplasm
4 TphA3 1
5 TphA3 2
6 TphA3 3
7 TphA3 4
8 TphA3 5
9 TphA3 6
10 TphA3 7
11 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]-

Purification of TphA1

A
Fractions of the TphA1 purfication
Band Sample Fraction
1 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]
2 TphA1 1
3 TphA1 2
4 TphA1 3
5 TphA1 4
6 TphA1 5
7 TphA1 6
  • Note: The other bands over TphA1 represent a contamination by aroY

Purification of TphB

A
Fractions of the TphB purfication
Band Sample Fraction
1 TphA1 1
2 TphA1 2
3 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]
4 TphA1 3
5 TphA1 4
6 TphA1 5
7 TphA1 6
7 TphA1 7

Purification of TphA2

  • Note: We didn't perform an SDS-Page from the purification.

week 19 (17.-21.09.12)

Operon Construction

A
Test restriction of J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB, J61002-aroY and BBa_K316003 (far left: Lambda DNA/Eco47I (AvaII) Marker, 13)
  • J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB showed unexpected bands although only one was expected after cutting with SpeI and PstI
  • BBa_K316003 showed two bands when cut with PstI and SpeI and PstI respectively where only one was expected
  • This lead us to the assumption that our PstI enzyme was contaminated with EcoRI (note the slightly differences between BBa_K316003 cut with SpeI and PstI and BBa_K316003 cut with XbaI and PstI)
  • Nevertheless also J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB seemed to possess mutation(s) introducing new recognition sites for SpeI and/or PstI
  • As we lacked time to confirm our assumptions by sequencing we decided to cancel further operon construction

week 20 (24.-26.09.12)

Enzyme assays

  • TpH enzymes
    • We mesure no activity
  • AroY
    • For protocol see here
    • Bevor addition of XylE we see a brown solution. The brown color is typical for 1,2-Benzoquinone.
    • After the addition of XylE we see a very high activity.
A
Funktional test for AroY: 1) AroY with 50 mM Protocatechuate after 24 h; 2) 1) after addition of XylE and 10 min inkubation
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