Team:TU Darmstadt/Labjournal/Metabolism

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 DSMZ-German Collection of Microorganism and Cell Cultures (DSMZ no.[http://www.dsmz.de/catalogues/details/culture/DSM-14576.html?tx_dsmzresources_pi5%5BreturnPid%5D=304 14576]). For more informations you will see our project discription.

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

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

tphB (both single bands at 1000 bp) isolated with colony PCR from C. testosteroni KF-1 genome (far left: 1kb DNA ladder, NEB)

Other

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

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

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

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

Plamid backbone	Concentration [ng/µl]
pSB1C3	42.6

restriction of BBa_K316003 using EcoRI and PstI (1kb DNA ladder, NEB)

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

Insert	Concentration [ng/µl]
xylE-dT	22.2

week 5 (11.-15-06.12)

Other

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

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-media-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-media-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:

• 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.

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

• Restriction 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

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

• Restriction 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-media-chloramphenicol with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction 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

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

• Restriction 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-media-chloramphenicol with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction 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

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)

• Inoculation of 10 mL of LB-media-chloramphenicol with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

Miniprep	Concentration [ng/µl]
pSB1C3-tphA1	60.5

• Restriction of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI

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-media-kanamycin with one colony of the transformation and incubation
• Miniprep of the culture

Miniprep	Concentration [ng/µl]
tphA2 gene synthesis	112.6

• 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

Colony PCR on psB1C3-tphA2. From left to right: colony 1-12 (far right: BenchTop 1kb DNA ladder, Promega)

• Inoculation of 10 mL of LB-media-chloramphenicol with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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-media-kanamycin with one colony of the transformation and incubation
• Miniprep of the culture

Miniprep	Concentration [ng/µl]
aroY gene synthesis	63.25

• 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 Nanodrop

Plamid backbone	Concentration [ng/µl]
J61002	42.5

week 15 (20.-24.08.12)

Other

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

Midiprep	Concentration [ng/µl]
pPR-IBA2	127

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

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

• Restriction of RBS-tphA1 with EcoRI and PstI
• Ligation of RBS-tphA1 (cut with EcoRI and PstI) and J61002 (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-media-ampicillin with colony 4 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction of RBS-tphA2 with EcoRI and PstI
• Ligation of RBS-tphA2 (cut with EcoRI and PstI) and J61002 (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-media-ampicillin with colony 6 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

Miniprep	Concentration [ng/µl]
J61002-RBS-tphA2	80.3

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

• Restriction of RBS-tphA3 with EcoRI and PstI
• Ligation of RBS-tphA3 (cut with EcoRI and PstI) and J61002 (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-media-ampicillin with colony 6 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction of RBS-tphB with EcoRI and PstI
• Ligation of RBS-tphB (cut with EcoRI and PstI) and J61002 (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-media-ampicillin with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction of RBS-aroY with EcoRI and PstI
• Ligation of RBS-aroY (cut with EcoRI and PstI) and J61002 (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-media-ampicillin with colony 2 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

Miniprep	Concentration [ng/µl]
J61002-RBS-aroY	77.2

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

• Restriction of tphA1_over-ex with EcoRI and PstI
• Ligation of tphA1_over-ex (cut with EcoRI and PstI) and pPR-IBA2 (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-media-ampicillin with colony 3 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction of tphA2_over-ex with EcoRI and PstI
• Ligation of tphA2_over-ex (cut with EcoRI and PstI) and pPR-IBA2 (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-media-ampicillin with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction of tphA3_over-ex with EcoRI and PstI
• Ligation of tphA3_over-ex (cut with EcoRI and PstI) and pPR-IBA2 (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-media-ampicillin with colony 4 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction of tphB_over-ex with EcoRI and PstI
• Ligation of tphB_over-ex (cut with EcoRI and PstI) and pPR-IBA2 (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-media-ampicillin with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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

• Restriction of aroY_over-ex with EcoRI and PstI
• Ligation of aroY_over-ex (cut with EcoRI and PstI) and pPR-IBA2 (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-media-ampicillin with colony 3 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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
  • Concentrations measured by Nanodrop

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

Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-4 (far right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)

• Inoculation of 10 mL of LB-media-ampicillin with colony 4 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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
  • Concentrations measured by Nanodrop

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

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)

• Inoculation of 10 mL of LB-media-ampicillin with colony 1 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

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
  • Concentrations measured by Nanodrop

Insert	Concentration [ng/µl]
RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI)	129.5

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

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)

• Inoculation of 10 mL of LB-media-ampicillin with colony 2 and incubation
• Miniprep of the culture and a glycerine stock was made
• Concentrations measured by Nanodrop

Miniprep	Concentration [ng/µl]
J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB	312.2

Overexpression

tphA2

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

tphB

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

aroY

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

tphA1

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

tphA3

• Inoculate 10 mL of LB-media-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

Results of the overexpression tphA2/tphB

SDS-PAGE of overexpression from all five genes

• SDS-Page according to standard protocol

Results of the overexpression aroY/tphA3/tphA1/tphA2/tphB/

week 18 (10.-17.09.12)

Purification of aroY

• Protein purification according to standard strep-tag purification protocol

Fractions of the aroY purfication

Purification of TphA3

• Protein purification according to standard strep-tag purification protocol

Fractions of the TphA3 purfication

Purification of TphA1

• Protein purification according to standard strep-tag purification protocol

Fractions of the TphA1 purfication

• Note: The other bands over TphA1 represent a contamination by aroY

Purification of TphB

• Protein purification according to standard strep-tag purification protocol

Fractions of the TphB purfication

Purification of TphA2

• Protein purification according to standard strep-tag purification protocol

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

week 19 (17.-21.09.12)

Gel permeation chromatography

• We performed the GPC with the following conditions:

• We injected 50 µl of fraction 3 from each protein (TphA1, TphA2, TphA3, TphB and AroY) for analysis of the molar mass and the oligomerization.
  • Results:

GPC analysis of TphA3. The Peak of TphA3 has a retention time of 33 minutes. This retention time corresponds to a molar mass of 52 kDa, approximately.

GPC analysis of TphB. The Peak of TphB has a retention time of 32.5 minutes. This retention time corresponds to a molar mass of 64 kDa, approximately. The peak at minute 13 is an undefined contamination.

GPC analysis of AroY. The Peak of AroY has a retention time of 28 minutes. This retention time corresponds to a molar mass of 285.4 kDa, approximately.

• Note: The GPC analysis of TphA2 and TphA1 respectively didn't work. The peak at 39 minutes is desthiobiotin from the protein purification.

Operon Construction

• After several failed cloning attempts a test restriction digest was performed

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
  • For the protocol, see here
  • We didn't measure the activity.

• AroY
  • For the protocol, see here
  • Before the addition of XylE we observed a brown solution. The brown color is typical for 1,2-Benzoquinone.
  • After the addition of XylE we observed a very high activity.

Functional test for AroY:
1) AroY with 50 mM Protocatechuate after 24 h;
2) after addition of [http://partsregistry.org/Part:BBa_K316003 XylE] and 10 min incubation