Team:TU Darmstadt/Labjournal/Metabolism

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Latest revision as of 02:10, 27 September 2012

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:

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.

[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

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

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

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

Protein purification according to standard strep-tag purification protocol

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

Protein purification according to standard strep-tag purification protocol

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

Protein purification according to standard strep-tag purification protocol

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

Protein purification according to standard strep-tag purification protocol

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

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:

Instrument	Pharmacia FPLC System
Colum	Superose 6 10/30
Mobile phase	PBS pH 7.4
Detector	280 nm
Flow rate	0.5 ml/min
Progam	Isocatic

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

@@ Line 1: / Line 1: @@
-===week 1 (14.-18.05.12)===
+<html>
-'''tphA1'''
+<link rel="stylesheet" href="https://2012.igem.org/wiki/index.php?title=Team:TU_Darmstadt/css&amp;action=raw&amp;ctype=text/css" type="text/css" />
-* No work progress
+<div id="TUD">
-'''tphA2'''
+<div id="top">
-* No work progress
+	<ul>
-'''tphA3'''
+		<li><a href="http://www.igem.tu-darmstadt.de/igem/projekt/index.en.jsp" title="iGEM">iGEM@TUD</a></li>
-* No work progress
+		<li><a href="/Team:TU_Darmstadt" title="Home">Home</a></li>
-'''tphB'''
+		<li><a href="/Team:TU_Darmstadt/Project" title="Project">Project</a><ul>
-* No work progress
+	        <li><a href="/Team:TU_Darmstadt/Project" title="Team:Overview">Overview</a></li>
-'''aroY'''
+	        <li><a href="/Team:TU_Darmstadt/Project/Degradation" title="Degradation">1. Degradation</a></li>
-* No work progress
+	        <li><a href="/Team:TU_Darmstadt/Project/Transport" title="Transport">2. Transport</a></li>
+	        <li><a href="/Team:TU_Darmstadt/Project/Metabolism" title="Metabolism">3. Metabolism</a></li>
+	        <li><a href="/Team:TU_Darmstadt/Project/Material_Science" title="Material Science">4. Material Science</a></li>
+	        <li><a href="/Team:TU_Darmstadt/Project/Simulation" title="Simulation">5. Simulation</a></li>
+	        <li><a href="/Team:TU_Darmstadt/Project/Ecology" title="Ecology">Ecology</a></li>
+	        <li><a href="/Team:TU_Darmstadt/Project/Philosophy" title="Philosophy">Philosophy</a></li></ul></li>
+		<li><a href="/Team:TU_Darmstadt/Team" title="Team:TU_Darmstadt/Team">Team</a><ul>
+	        <li><a href="/Team:TU_Darmstadt/Team" title="Team:TU_Darmstadt/Team">Members</a></li>
+	        <li><a href="/Team:TU_Darmstadt/Supporters" title="Team:TU_Darmstadt/Supporters">Supporters</a></li></ul></li>
+		<li><a href="/Team:TU_Darmstadt/Parts" title="BioBricks">BioBricks</a></li>
+		<li><a href="/Team:TU_Darmstadt/Documentation" title="Documentation">Documentation</a><ul>
+			<li><a href="/Team:TU_Darmstadt/Documentation" title="Documentation">Overview</a></li>
+		    <li><a href="/Team:TU_Darmstadt/Labjournal" title="Labjournal">Labjournal</a></li>
+		    <li><a href="/Team:TU_Darmstadt/Protocols" title="Protocols">Protocols</a></li>
+			<li><a href="/Team:TU_Darmstadt/Materials" title="Materials">Materials</a></li>
+			<li><a href="/Team:TU_Darmstadt/Modeling" title="Modeling">Modeling</a></li>
+		    <li><a href="/Team:TU_Darmstadt/Safety" title="Safety">Safety</a></li>
+		    <li><a href="/Team:TU_Darmstadt/Downloads" title="Downloads">Downloads</a></li></ul></li>
+		<li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a></li>
+		<li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Sponsors</a><ul>
+		    <li><a href="/Team:TU_Darmstadt/Sponsors" title="Sponsors">Overview</a></li>
+		    <li><a href="/Team:TU_Darmstadt/Contact" title="Contact">Benefits</a></li></ul></li>
+	</ul>
+<div id="igem"><a href="https://2012.igem.org/Main_Page"></a></div>
+</div>
+<!-- end #menu -->
+</html>
+<span style="font-size:200%;"><span style="color:#00689D;">Labjournal Metabolism</span></span>
+This lab journal describes a isolation and characterisation of the terephthalic acid 1,2-dioxigenase system and an dihydrodiol decarboxylase from [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Bacteria 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 [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism project discription].
+[[File:Klonierung_sascha.png|900px]]
+==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]]
+* [[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
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Production_of_chemically_competent_cells Production of chemically ] competent ''E. coli DH5α'' and ''E. coli BL21(DE3)pLysS'' cells
-===week 2 (21.-25.05.12)===
+==week 2 (21.-25.05.12)==
 '''tphA1'''
-* Isolation of the gene from ''C. testosteroni KF-1'' genome using [[colony-PCR]]
+* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR]
 ** Annealing temperature: 49 °C
-** [[Primer]]: tphA1-l-F and tphA1-l-R
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-l-F and tphA1-l-R
+** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+::{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA1 || 0.6
+|}
-'''tphA2'''
-* No work progress
 '''tphA3'''
-* Isolation of the gene from ''C. testosteroni KF-1'' genome using [[colony-PCR]]
+* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR]
 ** Annealing temperature: 59 °C
-** [[Primer]]: tphA3-l-F and tphA3-l-R
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA3-l-F and tphA3-l-R
+** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+::{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA3 || 0.1
+|}
+[[File:WIKI-2012-05-23_colony_PCR_cTestosteroni_A1_A3.png|thumb|none|alt=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]]
+* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR]
 ** Annealing temperature: 59 °C
-** [[Primer]]: tphB-l-F and tphB-l-R
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphB-l-F and tphB-l-R
-'''aroY'''
+** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-* No work progress
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+::{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphB || 0.1
+|}
+[[File:WIKI-2012-05-23_colony_PCR_cTestosteroni_B.png|thumb|none|alt=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'''
-* [[Transformation]] and [[midi prep]] of all used [[Biobricks]]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Plasmid_Midiprep midi prep] of all used [[Biobricks]]
-** Concentrations measured by [[Nanoprop]]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 ::{| class="wikitable"
 |-
 ! Biobrick!! Concentration [ng/µl]
 |-
-| BBa_K316003 || -
+| BBa_K316003 || 114.9
 |-
-| BBa_J23100 || -
+| BBa_J23100 || 450.2
 |-
-| BBa_B0015 || -
+| BBa_B0015 || 314.1
 |-
-| BBa_J61101 || -
+| BBa_J61101 || 86.1
 |}
-===week 3 (28.05.-01.06.12)===
+==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''
+** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphA1 isolated from ''C. testosteroni''
 *** Annealing temperature: 69 °C
-*** [[Primer]]: tphA1-l-PstI(99)-R and tphA1-l-R
+*** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-l-PstI(99)-R and tphA1-l-R
 ** tphA1 fragment 2
-** [[PCR]] on tphA1 isolated from ''C. tesosteroni''
+** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphA1 isolated from ''C. testosteroni''
 *** Annealing temperature: 69 °C
-*** [[Primer]]: tphA1-l-PstI(99)-F and tphA1-l-F
+*** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-l-PstI(99)-F and tphA1-l-F
-** Both PCR products were purified via [[gel extraction]]
+** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-** Concentrations measured by [[Nanoprop]]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 ::{| class="wikitable"
 |-
 ! PCR product!! Concentration [ng/µl]
 |-
-| Fragment 1 || -
+| Fragment 1 || 40.3
 |-
-| Fragment 2 || -
+| Fragment 2 || 62.1
 |}
-* Both fragments were cut with BsaI in a [[restriction digest]]
+[[File:WIKI-2012-05-29_tphA1_mutation.jpg|thumb|none|alt=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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction]
 ** The ligation mix differed from our standard protocol in the following manner
 *** 100 ng of fragment 1
@@ Line 79: / Line 144: @@
 *** add DI water up to 20 µL
 *** incubate for 15 minutes at 37 °C
-** [[PCR]] on ligation mix
+** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on ligation mix
 *** Annealing temperature: 59 °C
-*** [[Primer]]: tphA1-l-R and tphA1-l-F
+*** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-l-R and tphA1-l-F
-** The PCR product was purified via [[gel extraction]]
+** The PCR product was purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-** Concentrations measured by [[Nanoprop]]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 ::{| class="wikitable"
 |-
 ! PCR product!! Concentration [ng/µl]
 |-
-| Mutated tphA1 || -
+| Mutated tphA1 || 86.1
 |}
-'''tphA2'''
-* No work progress
+==week 4 (04.-08.06.12)==
+'''Other'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_K316003 by EcoRI and PstI
+** Purification of plasmid backbone pSB1C3 via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+::{| class="wikitable"
+|-
+! Plamid backbone!! Concentration [ng/µl]
+|-
+| pSB1C3 || 42.6
+|}
+[[File:WIKI-2012-06-06-_xylE_plasmid_restriction.jpg|thumb|none|alt=A|restriction  of BBa_K316003 using EcoRI and PstI (1kb DNA ladder, NEB)]]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_K316003 by XbaI and PstI
+** Purification of insert xylE-dT via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+::{| class="wikitable"
+|-
+! Insert!! Concentration [ng/µl]
+|-
+| xylE-dT || 22.2
+|}
+==week 5 (11.-15-06.12)==
+'''Other'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_J23100 by SpeI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the restriction
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
+** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
+* After overnight incubation of colony 2 in [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media] with ampicilin a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB 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:
+{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
+|- style="font-size:11pt"  valign="bottom"
+| width="60" height="15" |''' Nummer'''
+| width="120" |''' Standard [mM]'''
+|- style="font-size:11pt"  valign="bottom"
+| align="center" height="15" | 1
+| align="center" | 1
+|- style="font-size:11pt"  valign="bottom"
+| align="center" height="15" | 2
+| align="center" | 2
+|- style="font-size:11pt"  valign="bottom"
+| align="center" height="15" | 3
+| align="center" | 5
+|- style="font-size:11pt"  valign="bottom"
+| align="center" height="15" | 4
+| align="center" | 10
+|- style="font-size:11pt"  valign="bottom"
+| align="center" height="15" | 5
+| align="center" | 12.5
+|- style="font-size:11pt"  valign="bottom"
+| align="center" height="15" | 6
+| align="center" | 15
+|- style="font-size:11pt"  valign="bottom"
+| align="center" height="15" | 7
+| align="center" | 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.
+[[File:Bild3ddd.png|550px| Michaelis Menten plot of XylE with protocatechuic acid as substrate ]]
+{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
+|- style="font-size:11pt;font-weight:bold" align="center" valign="bottom"
+| width="134" height="15" | [Protocatecuatuic acid] mM
+| width="124" | Velocity units per minute
+|- style="font-size:11pt" align="center" valign="bottom"
+| align="center" height="15" | 1
+| align="center" | 0
+|- style="font-size:11pt" align="center" valign="bottom"
+| align="center" height="15" | 2
+| align="center" | 0
+|- style="font-size:11pt" align="center" valign="bottom"
+| align="center" height="15" | 5
+| align="center" | 0
+|- style="font-size:11pt" align="center" valign="bottom"
+| align="center" height="15" | 10
+| align="center" | 0
+|- style="font-size:11pt" align="center" valign="bottom"
+| align="center" height="15" | 12,5
+| align="center" | 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'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on mutated tphA1
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-Suffix_R and tphA1-l-Prefix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| Mutated tphA1-prefix/suffix || 62.0
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was negative
 '''tphA3'''
-* No work progress
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphA3 isolated from ''C. testosteroni''
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA3-Prefix_F and tphA3-Suffix_R
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA3-prefix/suffix || 30.5
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA3-prefix/suffix with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA3-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pSB1C3-tphA3-prefix/suffix || 79.6
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
+* Preparation for sequencing
+** Sequence was confirmed
 '''tphB'''
-* No work progress
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphB isolated from ''C. testosteroni''
-'''aroY'''
+** Annealing temperature: 59 °C
-* No work progress
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphB-Prefix and tphB-Suffix_R
-'''Other'''
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphB_prefix/suffix || 20.3
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was negative
-===week 4 (04.-08.06.12)===
+==week 11 (23.-27.07.12)==
+'''tphB'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphB isolated from ''C. testosteroni''
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphB-Prefix and tphB-Suffix_R
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphB_prefix/suffix || 52.5
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pSB1C3-tphB-prefix/suffix || 35.8
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
+* Preparation for sequencing
+** Sequence was confirmed
+==week 12 (30.07.-03.08.12)==
 '''tphA1'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on mutated tphA1
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-Suffix_R and tphA1-l-Prefix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| Mutated tphA1-prefix/suffix || 34.2
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+[[File:WIKI-2012-07-31_pSB1C3-tphA1_colony_PCR.jpg|thumb|none|alt=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)]]
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pSB1C3-tphA1 || 60.5
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
+[[File:WIKI-2012-08-02_pSB1C3-tphA1_test_restriction.jpg|thumb|none|x300px|alt=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
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the tphA2 gene synthesis
+* Inoculation of 10 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-kanamycin with one colony of the transformation and incubation
+* [[Miniprep]] of the culture
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| tphA2 gene synthesis || 112.6
+|}
+*[[Restriktion digest]] of the tphA2 gene synthesis with EcoRI and PstI
+*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the tphA2 gene synthesis and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
-'''tphA3'''
+[[File:WIKI-2012-08-07_pSB1C3-tphA2_colony_PCR.jpg|thumb|none|alt=A|Colony PCR on psB1C3-tphA2. From left to right: colony 1-12 (far right: BenchTop 1kb DNA ladder, Promega)]]
-'''tphB'''
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pSB1C3-tphA2-prefix/suffix || 111.1
+|}
+* Preparation for sequencing
+** Sequence was confirmed
-'''aroY'''
+==week 14 (13.-17.08.12)==
+'''aroY'''
+* Reconstitution of the aroY gene synthesis
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the aroY gene synthesis
+* Inoculation of 10 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-kanamycin with one colony of the transformation and incubation
+* [[Miniprep]] of the culture
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| aroY gene synthesis || 63.25
+|}
+*[[Restriktion digest]] of the aroY gene synthesis with EcoRI and PstI
+*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the aroY gene synthesis and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was negative
 '''Other'''
-* [[Restriction]] digest of BBa_K316003 by EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_J23100 by EcoRI and PstI
-** Purification of plasmid backbone pSB1C3 via [[gel extraction]]
+** Purification of plasmid backbone J61002 via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-** Concentrations measured by [[Nanoprop]]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 ::{| class="wikitable"
 |-
 ! Plamid backbone!! Concentration [ng/µl]
 |-
-| pSB1C3 || -
+| J61002 || 42.5
 |}
-* [[Restriction]] digest of BBa_K316003 by XbaI and PstI
-** Purification of insert xylE-dT via [[gel extraction]]
+==week 15 (20.-24.08.12)==
-** Concentrations measured by [[Nanoprop]]
+'''Other'''
+* Designing primers for over expression and operon construction
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of pPR-IBA2
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with one colony and incubation
+* [[Midiprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Midiprep!! Concentration [ng/µl]
+|-
+| pPR-IBA2 || 127
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of pPR-IBA2 with EcoRI and PstI
+** Purification of plasmid backbone pPR-IBA2 via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 ::{| class="wikitable"
 |-
 ! Plamid backbone!! Concentration [ng/µl]
 |-
-| xylE-dT || -
+| pPR-IBA2 || 35.6
 |}
-===week 5 (11.-15-06.12)===
+==week 16 (27.-31.08.12)==
+===Operon construction===
 '''tphA1'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA1
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: RBS-tphA1 and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA1 with RBS || 33.5
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphA1 with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA1 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 4 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-tphA1 || 79,6
+|}
 '''tphA2'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA2
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: RBS-tphA2 and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA2 with RBS || 46.8
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphA2 with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA2 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 6 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-tphA2 || 80.3
+|}
-'''tphA3'''
+[[File:WIKI-2012-08-29_Colony_PCR_RBS_A1_RBS_A2.png|thumb|none|alt=A|Colony PCR on J61002-RBS-tphA1 and J61002-RBS-tphA2 (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
+'''tphA3'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA3
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: RBS-tphA3 and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA3 with RBS || 26.5
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphA3 with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA3 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 6 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-tphA3 || 67.5
+|}
 '''tphB'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphB
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: RBS-tphB and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphB with RBS || 49.2
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphB with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphB (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-tphB || 65.8
+|}
+'''aroY'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-aroY
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: RBS-aroY and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| aroY with RBS || 55.2
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-aroY with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-aroY (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 2 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-aroY || 77.2
+|}
+[[File:WIKI-2012-08-29_Colony_PCR_RBS_A3_RBS_B_RBS_aroY.png|thumb|none|alt=A|Colony PCR on J61002-RBS-tphA3, J61002-RBS-tphB and J61002-RBS-aroY (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
+===Over expression===
+'''tphA1'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA1
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphA1 and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA1_over-ex || 116.2
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA1_over-ex with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA1_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 3 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pPR-IBA2-tphA1_over-ex || 98.5
+|}
+'''tphA2'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA2
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphA2 and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA2_over-ex || 63.9
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA2_over-ex with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA2_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pPR-IBA2-tphA2_over-ex || 85.2
+|}
+'''tphA3'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA3
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphA3 and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphA3_over-ex || 90.4
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA3_over-ex with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA3_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 4 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pPR-IBA2-tphA3_over-ex || 85.9
+|}
+'''tphB'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphB
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphB and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| tphB_over-ex || 87.5
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphB_over-ex with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphB_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pPR-IBA2-tphB_over-ex || 85.2
+|}
 '''aroY'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-aroY
+** Annealing temperature: 59 °C
+** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-aroY and Suffix
+* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! PCR product!! Concentration [ng/µl]
+|-
+| aroY_over-ex || 105.1
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of aroY_over-ex with EcoRI and PstI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of aroY_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
+** The PCR was positive
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 3 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| pPR-IBA2-aroY_over-ex || 92.1
+|}
-'''Other'''
+==week 17 (03.-07.09.12)==
-* [[Restriction]] digest of BBa_J23100 by SpeI and PstI
+===Operon construction===
-* [[Dephosphorylation]] of the restriction digest
+'''RBS-tphA1-RBS-tphA2'''
-* [[Ligation]] of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of J61002-RBS-tphA1 by EcoRI and SpeI
-** [[Transformation]] with 2 µL of the ligation mix
+* Purification of insert RBS-tphA1 RBS-tphA1 (cut with EcoRI and SpeI) via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-* [[Colony-PCR]] of the transformation for verification
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
-* After overnight [[incubation]] of colony x in [[LB medium]] with ampicilin a [[glycerine stock]] was made
+::{| class="wikitable"
+|-
+! Insert!! Concentration [ng/µl]
+|-
+| RBS-tphA1 (cut with EcoRI and SpeI)|| 50.2
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of J61002-RBS-tphA2 EcoRI and XbaI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)and RBS-tphA1 (cut with EcoRI and SpeI)
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
+** The PCR was positive
-===week 6 (18.-22.06.12)===
+[[File:WIKI-2012-09-03_colony_PCR_RBS-tphA1-RBS-tphA2.jpg|thumb|none|alt=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)]]
-* No work progress
-===week 7 (25.-29.06.12)===
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 4 and incubation
-'''tphA1'''
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-tphA1-RBS-tphA2 || 112.5
+|}
+'''RBS-tphA3-RBS-tphB'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of J61002-RBS-tphA3 by EcoRI and SpeI
+* Purification of insert RBS-tphA3 (cut with EcoRI and SpeI) via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+::{| class="wikitable"
+|-
+! Insert!! Concentration [ng/µl]
+|-
+| RBS-tphA3 (cut with EcoRI and SpeI)|| 178.9
+|}
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of J61002-RBS-tphB EcoRI and XbaI
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)and RBS-tphA3 (cut with EcoRI and SpeI)
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
+** The PCR was positive
-'''tphA2'''
+[[File:WIKI-2012-09-03_colony_PCR_Operon_A3-B.jpg|thumb|none|alt=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)]]
-'''tphA3'''
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-tphA3-RBS-tphB || 225.5
+|}
-'''tphB'''
+'''RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB'''
-'''aroY'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of J61002-RBS-tphA1-RBS-tphA2 by EcoRI and SpeI
+* Purification of insert RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI) via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
+** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+::{| class="wikitable"
+|-
+! Insert!! Concentration [ng/µl]
+|-
+| RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI)|| 129.5
+|}
-'''Other'''
+[[File:WIKI-2012-09-04_E%2BS_restriction_RBS_tphA1tphA2.jpg|thumb|none|alt=A|restriction  of of J61002-RBS-tphA1-RBS-tphA2 by EcoRI and SpeI (Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
-* 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)===
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of J61002-RBS-tphA3-RBS-tphB EcoRI and XbaI
-'''tphA1'''
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphA3-RBS-tphB (cut with EcoRI and XbaI)
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation 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)
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
+* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
+** The PCR was positive
-'''tphA2'''
+[[File:WIKI-2012-09-05_colony_PCR_Operon_A1-A2-A3-B.jpg|thumb|none|alt=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)]]
-'''tphA3'''
+* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 2 and incubation
+* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
+:{| class="wikitable"
+|-
+! Miniprep!! Concentration [ng/µl]
+|-
+| J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB || 312.2
+|}
+===Overexpression===
+'''tphA2'''
+* Inoculate 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
+* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
 '''tphB'''
+* Inoculate 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
+* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
 '''aroY'''
+* Inoculate 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
+* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
-'''Other'''
-===week 9 (09.-13.07.12)===
 '''tphA1'''
+* Inoculate 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
-'''tphA2'''
+* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
 '''tphA3'''
+* Inoculate 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
+* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
-'''tphB'''
+'''SDS-PAGE of tphB overexpression and tphA2 overexpression respectively'''
+* SDS-Page according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#SDS-PAGE protocol]
+[[File:TphB_tphA2_overex.jpg|thumb|none|alt=A|Results of the overexpression tphA2/tphB]]
+{| class="wikitable"
+|-
+! 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]||-
+|}
-'''aroY'''
+'''SDS-PAGE of overexpression from all five genes'''
+* SDS-Page according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#SDS-PAGE protocol]
+[[File: Overex_all.jpg |thumb|none|alt=A|Results of the overexpression aroY/tphA3/tphA1/tphA2/tphB/]]
+{| class="wikitable"
+|-
+! 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]||-
+|}
-'''Other'''
+==week 18 (10.-17.09.12)==
+'''Purification of aroY'''
+* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-===week 10 (16.-20.07.12)===
+[[File:Aro_purificate.jpg|thumb|none|alt=A|Fractions of the aroY purfication]]
-'''tphA1'''
-'''tphA2'''
+{| class="wikitable"
+|-
+! 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]||-
+|}
-'''tphA3'''
+'''Purification of TphA3'''
+* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-'''tphB'''
+[[File:Puri_A3.jpg|thumb|none|alt=A|Fractions of the TphA3 purfication]]
-'''aroY'''
+{| class="wikitable"
+|-
+! 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]||-
+|}
-'''Other'''
+'''Purification of TphA1'''
+* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-===week 11 (23.-27.07.12)===
+[[File:Puri_A1.jpg|thumb|none|alt=A|Fractions of the TphA1 purfication]]
-'''tphA1'''
-'''tphA2'''
+{| class="wikitable"
+|-
+! 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
+|-
+|}
-'''tphA3'''
+* Note: The other bands over TphA1 represent a contamination by aroY
-'''tphB'''
+'''Purification of TphB'''
+* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-'''aroY'''
+[[File:2012-09-26_18.07.04.jpg|thumb|none|alt=A|Fractions of the TphB purfication]]
-'''Other'''
+{| class="wikitable"
+|-
+! 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
+|-
+|}
-===week 12 (30.07.-03.08.12)===
+'''Purification of TphA2'''
-'''tphA1'''
+* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-'''tphA2'''
+* Note: We didn't perform an SDS-Page from the purification.
-'''tphA3'''
+==week 19 (17.-21.09.12)==
+===Gel permeation chromatography===
-'''tphB'''
+* We performed the GPC with the following conditions:
-'''aroY'''
+{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
+|- style="font-size:11pt" align="center" valign="bottom"
+| width="134" height="15" | Instrument
+| width="124" | Pharmacia FPLC System
-'''Other'''
+|- style="font-size:11pt" align="center" valign="bottom"
+| height="15" | Colum
+ | Superose 6 10/30
-===week 13 (06.-10.08.12)===
+|- style="font-size:11pt" align="center" valign="bottom"
-'''tphA1'''
+| height="15" | Mobile phase
+ | PBS pH 7.4
-'''tphA2'''
+|- style="font-size:11pt" align="center" valign="bottom"
+| height="15" | Detector
+ | 280 nm
-'''tphA3'''
+|- style="font-size:11pt" align="center" valign="bottom"
+| height="15" | Flow rate
+ | 0.5 ml/min
-'''tphB'''
+|- style="font-size:11pt" align="center" valign="bottom"
+| height="15" | Progam
+ | Isocatic
-'''aroY'''
+|}
-'''Other'''
+* 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:
+[[File:TphA3.JPG|550px|thumb|left|'''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.]]
+[[File:TphB.JPG|550px|thumb|left|'''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.]]
+[[File:AroY.JPG|550px|thumb|left|'''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.]]
-===week 14 (13.-17.08.12)===
-'''tphA1'''
-'''tphA2'''
-'''tphA3'''
-'''tphB'''
-'''aroY'''
-'''Other'''
-===week 15 (20.-24.08.12)===
-'''tphA1'''
-'''tphA2'''
-'''tphA3'''
-'''tphB'''
-'''aroY'''
-'''Other'''
-===week 16 (27.-31.08.12)===
-'''tphA1'''
-'''tphA2'''
-'''tphA3'''
-'''tphB'''
-'''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'''
+* 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction digest] was performed
+[[File:WIKI-2012-09-19_testrestriktion_a1-b_(s%2Bp)_aroy_(x%2Bp)_und_(von_links_nach_rechts)_s_p_s%2Bp_x%2Bp.jpg|thumb|none|alt=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'''
+**For the protocol, [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Enzyme_assays see here]
+**We didn't measure the activity.
+*'''AroY'''
+**For the protocol, [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Enzyme_assays 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.
+[[File:AroY_nachweiß.jpg|thumb|none|alt=A|'''Functional test for AroY:'''<br> '''1)''' AroY with 50 mM Protocatechuate after 24 h; <br>'''2)''' after addition of [http://partsregistry.org/Part:BBa_K316003 XylE] and 10 min incubation]]

Team:TU Darmstadt/Labjournal/Metabolism

From 2012.igem.org

Latest revision as of 02:10, 27 September 2012

Contents

week 1 (14.-18.05.12)

week 2 (21.-25.05.12)

week 3 (28.05.-01.06.12)

week 4 (04.-08.06.12)

week 5 (11.-15-06.12)

week 6 (18.-22.06.12)

week 7 (25.-29.06.12)

week 8 (02.-06.07.12)

week 9 (09.-13.07.12)

week 10 (16.-20.07.12)

week 11 (23.-27.07.12)

week 12 (30.07.-03.08.12)

week 13 (06.-10.08.12)

week 14 (13.-17.08.12)

week 15 (20.-24.08.12)

week 16 (27.-31.08.12)

Operon construction

Over expression

week 17 (03.-07.09.12)

Operon construction

Overexpression

week 18 (10.-17.09.12)

week 19 (17.-21.09.12)

Gel permeation chromatography

Operon Construction

week 20 (24.-26.09.12)

Enzyme assays