Team:TU Darmstadt/Labjournal/Metabolism

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Revision as of 10:55, 23 September 2012

Protocols Metabolism

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

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

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 x in LB medium with ampicilin a glycerine stock was made

week 6 (18.-22.06.12)

No work progress

week 7 (25.-29.06.12)

Other

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

week 8 (02.-06.07.12)

No work progress

week 9 (09.-13.07.12)

Other

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

week 10 (16.-20.07.12)

tphA1

PCR on mutated tphA1
- Annealing temperature: 59 °C
- Primer: tphA1-Suffix_R and tphA1-l-Prefix
Both PCR products were purified via gel extraction
Concentrations measured by 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-medium-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-medium-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-medium-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 restriciton digest of psB1C3-tphA1 with EcoRI and PstI (GeneRuler 100bp Plus DNA Ladder, Fermentas)

Preparation for sequencing
- Sequence was confirmed

week 13 (06.-10.08.12)

tphA2

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

Miniprep	Concentration [ng/µl]
tphA2 gene synthesis	112.6

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

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-medium-ampicillin with colony XXX 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-medium-ampicillin with colony XXX 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-medium-ampicillin with colony XXX 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

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

tphB

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

aroY

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

tphA1

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

tphA3

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

SDS-PAGE of tphB overexpression and tphA2 overexpression respectively

SDS-Page according to standard protocol

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 of aroY

week 19 (17.-21.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

@@ Line 47: / Line 47: @@
 '''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]
 * [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

Team:TU Darmstadt/Labjournal/Metabolism

From 2012.igem.org

Revision as of 10:55, 23 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)