Team:TU Darmstadt/Labjournal/Metabolism

From 2012.igem.org

(Difference between revisions)
(→week 3 (28.05.-01.06.12))
(→week 3 (28.05.-01.06.12))
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===week 3 (28.05.-01.06.12)===
===week 3 (28.05.-01.06.12)===
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on tphA1 isolated from ''C. tesosteroni''  
+
* 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
-
** Annealing temperature: 69 °C
+
** tphA1 fragment 1
-
** [[Primer]]: tphA1-l-PstI(99)-R and tphA1-l-R
+
** [[PCR]] on tphA1 isolated from ''C. tesosteroni''  
-
* [[PCR]] on tphA1 isolated from ''C. tesosteroni''  
+
*** Annealing temperature: 69 °C
-
** Annealing temperature: 69 °C
+
*** [[Primer]]: tphA1-l-PstI(99)-R and tphA1-l-R
-
** [[Primer]]: tphA1-l-PstI(99)-F and tphA1-l-F
+
** tphA1 fragment 2
 +
** [[PCR]] on tphA1 isolated from ''C. tesosteroni''  
 +
*** Annealing temperature: 69 °C
 +
*** [[Primer]]: tphA1-l-PstI(99)-F and tphA1-l-F
 +
** Both PCR products were purified via [[gel extraction]]
 +
::{| class="wikitable"
 +
|-
 +
! PCR product!! Concentraion [ng/µl]
 +
|-
 +
| Fragment 1 || -
 +
|-
 +
| Fragment 2 || -
 +
|}
 +
 
 +
* Both fragments were cut with BsaI in a [[restriction digest]]
 +
** The ligation mix differed from our standard protocol in the following manner
 +
*** 100 ng of fragment 1
 +
*** 200 ng of fragment 2
 +
*** 2 µL of 10x reaction buffer
 +
*** 1 µL of T4 DNA ligase
 +
*** add DI water up to 20 µL
 +
*** incubate for 15 minutes at 37 °C
 +
** [[PCR]] on ligation mix
 +
*** Annealing temperature: 59 °C
 +
*** [[Primer]]: tphA1-l-R and tphA1-l-F
 +
** The PCR product was purified via [[gel extraction]]
 +
::{| class="wikitable"
 +
|-
 +
! PCR product!! Concentraion [ng/µl]
 +
|-
 +
| Mutated tphA1 || -
 +
|}
'''tphA2'''
'''tphA2'''
* No work progress  
* No work progress  

Revision as of 09:51, 9 September 2012

Contents

week 1 (14.-18.05.12)

tphA1

  • No work progress

tphA2

  • No work progress

tphA3

  • No work progress

tphB

  • No work progress

aroY

  • No work progress

Other

  • Reconstitution of C. testosteroni KF-1 according to DSMZ [http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/engl_Opening.pdf protocol]
  • Cultivation of C. testosteroni KF-1 on agar plates with Medium 1
  • Production of chemically competent E. coli DH5α and E. coli BL21(DE3)pLysS cells

week 2 (21.-25.05.12)

tphA1

  • Isolation of the gene from C. testosteroni KF-1 genome using colony-PCR
    • Annealing temperature: 49 °C
    • Primer: tphA1-l-F and tphA1-l-R

tphA2

  • No work progress

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

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

aroY

  • No work progress

Other

Biobrick Concentraion [ng/µl]
BBa_K316003 -
BBa_J23100 -
BBa_B0015 -
BBa_J61101 -

week 3 (28.05.-01.06.12)

tphA1

  • Two PCRs were performed to mutate the PstI site in the wild type gene. The primer tphA1-l-PstI(99)-R and tphA1-l-PstI(99)-F respectively introduced a BsaI site
    • tphA1 fragment 1
    • PCR on tphA1 isolated from C. tesosteroni
      • Annealing temperature: 69 °C
      • Primer: tphA1-l-PstI(99)-R and tphA1-l-R
    • tphA1 fragment 2
    • PCR on tphA1 isolated from C. tesosteroni
      • Annealing temperature: 69 °C
      • Primer: tphA1-l-PstI(99)-F and tphA1-l-F
    • Both PCR products were purified via gel extraction
PCR product Concentraion [ng/µl]
Fragment 1 -
Fragment 2 -
  • Both fragments were cut with BsaI in a restriction digest
    • The ligation mix differed from our standard protocol in the following manner
      • 100 ng of fragment 1
      • 200 ng of fragment 2
      • 2 µL of 10x reaction buffer
      • 1 µL of T4 DNA ligase
      • add DI water up to 20 µL
      • incubate for 15 minutes at 37 °C
    • PCR on ligation mix
      • Annealing temperature: 59 °C
      • Primer: tphA1-l-R and tphA1-l-F
    • The PCR product was purified via gel extraction
PCR product Concentraion [ng/µl]
Mutated tphA1 -

tphA2

  • No work progress

tphA3

  • No work progress

tphB

  • No work progress

aroY

  • No work progress

Other

week 4 (04.-08.06.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 5 (11.-15-06.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 6 (18.-22.06.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 7 (25.-29.06.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 8 (02.-06.07.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 9 (09.-13.07.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 10 (16.-20.07.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 11 (23.-27.07.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 12 (30.07.-03.08.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

week 13 (06.-10.08.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

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

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