Team:TU Darmstadt/Labjournal/Metabolism

From 2012.igem.org

(Difference between revisions)
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* Reconstitution of ''C. testosteroni KF-1'' according to DSMZ [http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/engl_Opening.pdf protocol]
* 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 [[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'''
'''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
** Annealing temperature: 49 °C
** [[Primer]]: tphA1-l-F and tphA1-l-R
** [[Primer]]: tphA1-l-F and tphA1-l-R
-
** 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"
::{| class="wikitable"
|-
|-
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'''tphA3'''
'''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
** Annealing temperature: 59 °C
** [[Primer]]: tphA3-l-F and tphA3-l-R
** [[Primer]]: tphA3-l-F and tphA3-l-R
-
** 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"
::{| class="wikitable"
|-
|-
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'''tphB'''
'''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
** Annealing temperature: 59 °C
** [[Primer]]: tphB-l-F and tphB-l-R
** [[Primer]]: tphB-l-F and tphB-l-R
-
** 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"
::{| class="wikitable"
|-
|-
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'''Other'''
'''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"
::{| class="wikitable"
|-
|-
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* 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
* 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
** tphA1 fragment 1
-
** [[PCR]] on tphA1 isolated from ''C. testosteroni''  
+
** [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
*** Annealing temperature: 69 °C
*** [[Primer]]: tphA1-l-PstI(99)-R and tphA1-l-R
*** [[Primer]]: tphA1-l-PstI(99)-R and tphA1-l-R
** tphA1 fragment 2
** tphA1 fragment 2
-
** [[PCR]] on tphA1 isolated from ''C. testosteroni''  
+
** [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
*** Annealing temperature: 69 °C
*** [[Primer]]: tphA1-l-PstI(99)-F and tphA1-l-F
*** [[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"
::{| class="wikitable"
|-
|-
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-
* Both fragments were cut with BsaI in a [[restriction digest]]
+
* 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
** The ligation mix differed from our standard protocol in the following manner
*** 100 ng of fragment 1
*** 100 ng of fragment 1
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*** add DI water up to 20 µL
*** add DI water up to 20 µL
*** incubate for 15 minutes at 37 °C
*** 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
*** Annealing temperature: 59 °C
*** [[Primer]]: tphA1-l-R and tphA1-l-F
*** [[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"
::{| class="wikitable"
|-
|-
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==week 4 (04.-08.06.12)==
==week 4 (04.-08.06.12)==
'''Other'''
'''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_K316003 by EcoRI and PstI
-
** Purification of plasmid backbone pSB1C3 via [[gel extraction]]
+
** 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 [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
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|}
|}
-
[[File:WIKI-2012-06-06-_xylE_plasmid_restriction.jpg|thumb|none|alt=A|Restriction digest of BBa_K316003 using EcoRI and PstI (1kb DNA ladder, NEB)]]
+
[[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)]]
-
* [[Restriction]] digest of BBa_K316003 by XbaI and PstI
+
* [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 [[gel extraction]]
+
** 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 [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
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'''Other'''
'''Other'''
-
* [[Restriction]] digest of BBa_J23100 by SpeI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] digest of BBa_J23100 by SpeI and PstI
-
* [[Dephosphorylation]] of the restriction digest
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the restriction
-
* [[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#Ligation Ligation] of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
-
** [[Transformation]] of the ligation mix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
-
* [[Colony-PCR]] of the transformation for verification
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
* After overnight [[incubation]] of colony x in [[LB medium]] with ampicilin a [[glycerine stock]] was made
* After overnight [[incubation]] of colony x in [[LB medium]] with ampicilin a [[glycerine stock]] was made
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==week 10 (16.-20.07.12)==
==week 10 (16.-20.07.12)==
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on mutated tphA1  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on mutated tphA1  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: tphA1-Suffix_R and tphA1-l-Prefix
** [[Primer]]: tphA1-Suffix_R and tphA1-l-Prefix
-
* 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"
:{| class="wikitable"
|-
|-
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| Mutated tphA1-prefix/suffix || 62.0
| Mutated tphA1-prefix/suffix || 62.0
|}
|}
-
* [[Restriction digest]] of mutated tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was negative
** The PCR was negative
'''tphA3'''
'''tphA3'''
-
* [[PCR]] on tphA3 isolated from ''C. testosteroni''  
+
* [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
** Annealing temperature: 59 °C
** [[Primer]]: tphA3-Prefix_F and tphA3-Suffix_R
** [[Primer]]: tphA3-Prefix_F and tphA3-Suffix_R
-
* 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"
:{| class="wikitable"
|-
|-
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| tphA3-prefix/suffix || 30.5
| tphA3-prefix/suffix || 30.5
|}
|}
-
* [[Restriction digest]] of mutated tphA3-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphA3-prefix/suffix with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
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| pSB1C3-tphA3-prefix/suffix || 79.6
| pSB1C3-tphA3-prefix/suffix || 79.6
|}
|}
-
* [[Restriction digest]] of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
* Preparation for [[sequencing]]
* Preparation for [[sequencing]]
** Sequence was confirmed  
** Sequence was confirmed  
'''tphB'''
'''tphB'''
-
* [[PCR]] on tphB isolated from ''C. testosteroni''  
+
* [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
** Annealing temperature: 59 °C
** [[Primer]]: tphB-Prefix and tphB-Suffix_R
** [[Primer]]: tphB-Prefix and tphB-Suffix_R
-
* 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"
:{| class="wikitable"
|-
|-
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| tphB_prefix/suffix || 20.3
| tphB_prefix/suffix || 20.3
|}
|}
-
* [[Restriction digest]] of mutated tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was negative
** The PCR was negative
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'''tphB'''
'''tphB'''
-
* [[PCR]] on tphB isolated from ''C. testosteroni''  
+
* [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
** Annealing temperature: 59 °C
** [[Primer]]: tphB-Prefix and tphB-Suffix_R
** [[Primer]]: tphB-Prefix and tphB-Suffix_R
-
* 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"
:{| class="wikitable"
|-
|-
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| tphB_prefix/suffix || 52.5
| tphB_prefix/suffix || 52.5
|}
|}
-
* [[Restriction digest]] of mutated tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
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| pSB1C3-tphB-prefix/suffix || 35.8
| pSB1C3-tphB-prefix/suffix || 35.8
|}
|}
-
* [[Restriction digest]] of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
* Preparation for [[sequencing]]
* Preparation for [[sequencing]]
** Sequence was confirmed
** Sequence was confirmed
Line 307: Line 307:
==week 12 (30.07.-03.08.12)==
==week 12 (30.07.-03.08.12)==
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on mutated tphA1  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on mutated tphA1  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: tphA1-Suffix_R and tphA1-l-Prefix
** [[Primer]]: tphA1-Suffix_R and tphA1-l-Prefix
-
* 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"
:{| class="wikitable"
|-
|-
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| Mutated tphA1-prefix/suffix || 34.2
| Mutated tphA1-prefix/suffix || 34.2
|}
|}
-
* [[Restriction digest]] of mutated tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
Line 328: Line 328:
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 335: Line 335:
| pSB1C3-tphA1 || 60.5
| pSB1C3-tphA1 || 60.5
|}
|}
-
* [[Restriction digest]] of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
+
* [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 restriciton digest of psB1C3-tphA1 with EcoRI and PstI (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
[[File:WIKI-2012-08-02_pSB1C3-tphA1_test_restriction.jpg|thumb|none|x300px|alt=A|Test restriciton digest of psB1C3-tphA1 with EcoRI and PstI (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
Line 346: Line 346:
'''tphA2'''
'''tphA2'''
* Reconstitution of the tphA2 gene synthesis  
* Reconstitution of the tphA2 gene synthesis  
-
* [[Transformation]] 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 [[LB-medium]]-kanamycin with one colony of the transformation and [[incubation]]
* Inoculation of 10 mL [[LB-medium]]-kanamycin with one colony of the transformation and [[incubation]]
* [[Miniprep]] of the culture
* [[Miniprep]] of the culture
Line 356: Line 356:
|}
|}
*[[Restriktion digest]] of the tphA2 gene synthesis with EcoRI and PstI
*[[Restriktion digest]] of the tphA2 gene synthesis with EcoRI and PstI
-
*[[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony XX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony XX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 376: Line 376:
'''aroY'''
'''aroY'''
* Reconstitution of the aroY gene synthesis  
* Reconstitution of the aroY gene synthesis  
-
* [[Transformation]] 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 [[LB-medium]]-kanamycin with one colony of the transformation and [[incubation]]
* Inoculation of 10 mL [[LB-medium]]-kanamycin with one colony of the transformation and [[incubation]]
* [[Miniprep]] of the culture
* [[Miniprep]] of the culture
Line 386: Line 386:
|}
|}
*[[Restriktion digest]] of the aroY gene synthesis with EcoRI and PstI
*[[Restriktion digest]] of the aroY gene synthesis with EcoRI and PstI
-
*[[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was negative
** The PCR was negative
'''Other'''
'''Other'''
-
* [[Restriction]] digest of BBa_J23100 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 J61002 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"
::{| class="wikitable"
|-
|-
Line 405: Line 405:
'''Other'''
'''Other'''
* Designing primers for over expression and operon construction
* Designing primers for over expression and operon construction
-
* [[Transformation]] of pPR-IBA2  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of pPR-IBA2  
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with one colony and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with one colony and [[incubation]]
* [[Midiprep]] of the culture and a [[glycerine stock]] was made  
* [[Midiprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 415: Line 415:
| pPR-IBA2 || 127
| pPR-IBA2 || 127
|}
|}
-
* [[Restriction]] digest of pPR-IBA2 with EcoRI and PstI
+
* [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 [[gel extraction]]
+
** 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 [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
Line 428: Line 428:
===Operon construction===
===Operon construction===
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on pSB1C3-tphA1
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA1
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: RBS-tphA1 and Suffix
** [[Primer]]: RBS-tphA1 and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 439: Line 439:
| tphA1 with RBS || 33.5
| tphA1 with RBS || 33.5
|}
|}
-
* [[Restriction digest]] of RBS-tphA1 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphA1 with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 454: Line 454:
|}
|}
'''tphA2'''
'''tphA2'''
-
* [[PCR]] on pSB1C3-tphA2
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA2
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: RBS-tphA2 and Suffix
** [[Primer]]: RBS-tphA2 and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 465: Line 465:
| tphA2 with RBS || 46.8
| tphA2 with RBS || 46.8
|}
|}
-
* [[Restriction digest]] of RBS-tphA2 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphA2 with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 480: Line 480:
|}
|}
'''tphA3'''
'''tphA3'''
-
* [[PCR]] on pSB1C3-tphA3
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA3
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: RBS-tphA3 and Suffix
** [[Primer]]: RBS-tphA3 and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 491: Line 491:
| tphA3 with RBS || 26.5
| tphA3 with RBS || 26.5
|}
|}
-
* [[Restriction digest]] of RBS-tphA3 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphA3 with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 506: Line 506:
|}
|}
'''tphB'''
'''tphB'''
-
* [[PCR]] on pSB1C3-tphB
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphB
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: RBS-tphB and Suffix
** [[Primer]]: RBS-tphB and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 517: Line 517:
| tphB with RBS || 49.2
| tphB with RBS || 49.2
|}
|}
-
* [[Restriction digest]] of RBS-tphB with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphB with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 532: Line 532:
|}
|}
'''aroY'''
'''aroY'''
-
* [[PCR]] on pSB1C3-aroY
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-aroY
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: RBS-aroY and Suffix
** [[Primer]]: RBS-aroY and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 543: Line 543:
| aroY with RBS || 55.2
| aroY with RBS || 55.2
|}
|}
-
* [[Restriction digest]] of RBS-aroY with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-aroY with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 561: Line 561:
===Over expression===
===Over expression===
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on pSB1C3-tphA1
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA1
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: EcoRIGFxa-tphA1 and Suffix
** [[Primer]]: EcoRIGFxa-tphA1 and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 572: Line 572:
| tphA1_over-ex || 116.2
| tphA1_over-ex || 116.2
|}
|}
-
* [[Restriction digest]] of tphA1_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphA1_over-ex with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 587: Line 587:
|}
|}
'''tphA2'''
'''tphA2'''
-
* [[PCR]] on pSB1C3-tphA2
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA2
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: EcoRIGFxa-tphA2 and Suffix
** [[Primer]]: EcoRIGFxa-tphA2 and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 598: Line 598:
| tphA2_over-ex || 63.9
| tphA2_over-ex || 63.9
|}
|}
-
* [[Restriction digest]] of tphA2_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphA2_over-ex with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 613: Line 613:
|}
|}
'''tphA3'''
'''tphA3'''
-
* [[PCR]] on pSB1C3-tphA3
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA3
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: EcoRIGFxa-tphA3 and Suffix
** [[Primer]]: EcoRIGFxa-tphA3 and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 624: Line 624:
| tphA3_over-ex || 90.4
| tphA3_over-ex || 90.4
|}
|}
-
* [[Restriction digest]] of tphA3_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphA3_over-ex with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 639: Line 639:
|}
|}
'''tphB'''
'''tphB'''
-
* [[PCR]] on pSB1C3-tphB
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphB
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: EcoRIGFxa-tphB and Suffix
** [[Primer]]: EcoRIGFxa-tphB and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 650: Line 650:
| tphB_over-ex || 87.5
| tphB_over-ex || 87.5
|}
|}
-
* [[Restriction digest]] of tphB_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphB_over-ex with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 665: Line 665:
|}
|}
'''aroY'''
'''aroY'''
-
* [[PCR]] on pSB1C3-aroY
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-aroY
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
** [[Primer]]: EcoRIGFxa-aroY and Suffix
** [[Primer]]: EcoRIGFxa-aroY and Suffix
-
* 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"
:{| class="wikitable"
|-
|-
Line 676: Line 676:
| aroY_over-ex || 105.1
| aroY_over-ex || 105.1
|}
|}
-
* [[Restriction digest]] of aroY_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of aroY_over-ex with EcoRI and PstI
-
* [[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#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)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 694: Line 694:
===Operon construction===
===Operon construction===
'''RBS-tphA1-RBS-tphA2'''
'''RBS-tphA1-RBS-tphA2'''
-
* [[Restriction]] digest of J61002-RBS-tphA1 by EcoRI and SpeI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest 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]]
+
* 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]
-
** Concentrations measured by [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
Line 703: Line 703:
| RBS-tphA1 (cut with EcoRI and SpeI)|| 50.2
| RBS-tphA1 (cut with EcoRI and SpeI)|| 50.2
|}
|}
-
* [[Restriction digest]] of J61002-RBS-tphA2 EcoRI and XbaI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of J61002-RBS-tphA2 EcoRI and XbaI
-
* [[Dephosphorylation]] of the plasmid backbone J61002-RBS-tphA2 (cut with 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)
-
* [[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#Ligation 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
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
-
* [[Colony-PCR]] of the transformation for verification
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
** The PCR was positive
** The PCR was positive
Line 714: Line 714:
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony 4 and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony 4 and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 722: Line 722:
|}
|}
'''RBS-tphA3-RBS-tphB'''
'''RBS-tphA3-RBS-tphB'''
-
* [[Restriction]] digest of J61002-RBS-tphA3 by EcoRI and SpeI
+
* [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 [[gel extraction]]
+
* 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 [[Nanodrop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
Line 731: Line 731:
| RBS-tphA3 (cut with EcoRI and SpeI)|| 178.9
| RBS-tphA3 (cut with EcoRI and SpeI)|| 178.9
|}
|}
-
* [[Restriction digest]] of J61002-RBS-tphB EcoRI and XbaI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of J61002-RBS-tphB EcoRI and XbaI
-
* [[Dephosphorylation]] of the plasmid backbone J61002-RBS-tphB (cut with 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)
-
* [[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#Ligation 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
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
-
* [[Colony-PCR]] of the transformation for verification
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
** The PCR was positive
** The PCR was positive
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony XXX and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanodrop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 749: Line 749:
'''RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB'''
'''RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB'''
-
* [[Restriction digest]] of J61002-RBS-tphA1-RBS-tphA2 by EcoRI and SpeI
+
* [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 [[gel extraction]]
+
* 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 [[Nanodrop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
Line 759: Line 759:
|}
|}
-
[[File:WIKI-2012-09-04_E%2BS_restriction_RBS_tphA1tphA2.jpg|thumb|none|alt=A|Restriction digest of of J61002-RBS-tphA1-RBS-tphA2 by EcoRI and SpeI (Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
+
[[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)]]
-
* [[Restriction digest]] of J61002-RBS-tphA3-RBS-tphB EcoRI and XbaI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest 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)
+
* [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)
-
* [[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#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)
-
* [[Transformation]] of the ligation mix
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
-
* [[Colony-PCR]] of the transformation for verification
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
** The PCR was positive
** The PCR was positive
Line 772: Line 772:
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony 2 and [[incubation]]
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony 2 and [[incubation]]
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
-
* Concentrations measured by [[Nanodrop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
Line 784: Line 784:
'''tphA2'''
'''tphA2'''
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
-
* Over expression according to standard [[protocol]]  
+
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]  
'''tphB'''
'''tphB'''
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
-
* Over expression according to standard [[protocol]]
+
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
'''aroY'''
'''aroY'''
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
-
* Over expression according to standard [[protocol]]
+
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
'''tphA1'''
'''tphA1'''
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
-
* Over expression according to standard [[protocol]]
+
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
'''tphA3'''
'''tphA3'''
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* [[Inoculate]] 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
-
* Over expression according to standard [[protocol]]
+
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
'''SDS-PAGE of tphB overexpression and tphA2 overexpression respectively'''
'''SDS-PAGE of tphB overexpression and tphA2 overexpression respectively'''
-
* SDS-Page according to standard [[protocol]]  
+
* 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]]
[[File:TphB_tphA2_overex.jpg|thumb|none|alt=A|Results of the overexpression tphA2/tphB]]
{| class="wikitable"
{| class="wikitable"
Line 829: Line 829:
'''SDS-PAGE of overexpression from all five genes'''
'''SDS-PAGE of overexpression from all five genes'''
-
* SDS-Page according to standard [[protocol]]  
+
* 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/]]
[[File: Overex_all.jpg |thumb|none|alt=A|Results of the overexpression aroY/tphA3/tphA1/tphA2/tphB/]]
{| class="wikitable"
{| class="wikitable"
Line 862: Line 862:
==week 18 (10.-17.09.12)==
==week 18 (10.-17.09.12)==
'''Purification of aroY'''
'''Purification of aroY'''
-
* Protein purification according to [[standard strep-tag purification protocol]]
+
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
[[File:Aro_purificate.jpg|thumb|none|alt=A|Fractions of the aroY purfication]]
[[File:Aro_purificate.jpg|thumb|none|alt=A|Fractions of the aroY purfication]]
Line 882: Line 882:
'''Purification of TphA3'''
'''Purification of TphA3'''
-
* Protein purification according to [[standard strep-tag purification protocol]]
+
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
[[File:Puri_A3.jpg|thumb|none|alt=A|Fractions of the TphA3 purfication]]
[[File:Puri_A3.jpg|thumb|none|alt=A|Fractions of the TphA3 purfication]]
Line 914: Line 914:
'''Purification of TphA1'''
'''Purification of TphA1'''
-
* Protein purification according to [[standard strep-tag purification protocol]]
+
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
[[File:Puri_A1.jpg|thumb|none|alt=A|Fractions of the TphA1 purfication]]
[[File:Puri_A1.jpg|thumb|none|alt=A|Fractions of the TphA1 purfication]]

Revision as of 08:49, 23 September 2012

Protocols Metabolism


Contents

week 1 (14.-18.05.12)

Other

  • Reconstitution of C. testosteroni KF-1 according to DSMZ [http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/engl_Opening.pdf protocol]
  • Cultivation of C. testosteroni KF-1 on agar plates with 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

Biobrick Concentration [ng/µl]
BBa_K316003 114.9
BBa_J23100 450.2
BBa_B0015 314.1
BBa_J61101 86.1

week 3 (28.05.-01.06.12)

tphA1

  • Two PCRs were performed to mutate the PstI site in the wild type gene. The primer tphA1-l-PstI(99)-R and tphA1-l-PstI(99)-F respectively introduced a BsaI site
    • tphA1 fragment 1
    • PCR on tphA1 isolated from C. testosteroni
      • Annealing temperature: 69 °C
      • Primer: tphA1-l-PstI(99)-R and tphA1-l-R
    • tphA1 fragment 2
    • PCR on tphA1 isolated from C. testosteroni
      • Annealing temperature: 69 °C
      • Primer: tphA1-l-PstI(99)-F and tphA1-l-F
    • Both PCR products were purified via gel extraction
    • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
Fragment 1 40.3
Fragment 2 62.1
A
tphA1 Fragment 1 (left) and tphA1 Fragment 2 (right) after PCR (GeneRuler 100bp Plus DNA Ladder)


  • Both fragments were cut with BsaI in a restriction
    • The ligation mix differed from our standard protocol in the following manner
      • 100 ng of fragment 1
      • 200 ng of fragment 2
      • 2 µL of 10x reaction buffer
      • 1 µL of T4 DNA ligase
      • add DI water up to 20 µL
      • incubate for 15 minutes at 37 °C
    • PCR on ligation mix
      • Annealing temperature: 59 °C
      • Primer: tphA1-l-R and tphA1-l-F
    • The PCR product was purified via gel extraction
    • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
Mutated tphA1 86.1

week 4 (04.-08.06.12)

Other

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

week 5 (11.-15-06.12)

Other

week 6 (18.-22.06.12)

  • No work progress

week 7 (25.-29.06.12)

Other

  • 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

tphA3

  • PCR on tphA3 isolated from C. testosteroni
    • Annealing temperature: 59 °C
    • Primer: tphA3-Prefix_F and tphA3-Suffix_R
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA3-prefix/suffix 30.5
Miniprep Concentration [ng/µl]
pSB1C3-tphA3-prefix/suffix 79.6
  • restriction of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
  • Preparation for sequencing
    • Sequence was confirmed

tphB

  • PCR on tphB isolated from C. testosteroni
    • Annealing temperature: 59 °C
    • Primer: tphB-Prefix and tphB-Suffix_R
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphB_prefix/suffix 20.3

week 11 (23.-27.07.12)

tphB

  • PCR on tphB isolated from C. testosteroni
    • Annealing temperature: 59 °C
    • Primer: tphB-Prefix and tphB-Suffix_R
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphB_prefix/suffix 52.5
Miniprep Concentration [ng/µl]
pSB1C3-tphB-prefix/suffix 35.8
  • restriction of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
  • Preparation for sequencing
    • Sequence was confirmed

week 12 (30.07.-03.08.12)

tphA1

  • PCR on mutated tphA1
    • Annealing temperature: 59 °C
    • Primer: tphA1-Suffix_R and tphA1-l-Prefix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
Mutated tphA1-prefix/suffix 34.2
A
Colony PCR of pSB1C3-tphA1; from left to right: Colony 1-11 (BenchTop 1kb DNA ladder between colony 6 and 7 and on the far right)
Miniprep Concentration [ng/µl]
pSB1C3-tphA1 60.5
  • restriction of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
A
Test 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
Miniprep Concentration [ng/µl]
pSB1C3-tphA2-prefix/suffix 111.1
  • Preparation for sequencing
    • Sequence was confirmed

week 14 (13.-17.08.12)

aroY

Miniprep Concentration [ng/µl]
aroY gene synthesis 63.25

Other

Plamid backbone Concentration [ng/µl]
J61002 42.5

week 15 (20.-24.08.12)

Other

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

week 16 (27.-31.08.12)

Operon construction

tphA1

  • PCR on pSB1C3-tphA1
    • Annealing temperature: 59 °C
    • Primer: RBS-tphA1 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA1 with RBS 33.5
Miniprep Concentration [ng/µl]
J61002-RBS-tphA1 79,6

tphA2

  • PCR on pSB1C3-tphA2
    • Annealing temperature: 59 °C
    • Primer: RBS-tphA2 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA2 with RBS 46.8
Miniprep Concentration [ng/µl]
J61002-RBS-tphA2 80.3

tphA3

  • PCR on pSB1C3-tphA3
    • Annealing temperature: 59 °C
    • Primer: RBS-tphA3 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA3 with RBS 26.5
Miniprep Concentration [ng/µl]
J61002-RBS-tphA3 67.5

tphB

  • PCR on pSB1C3-tphB
    • Annealing temperature: 59 °C
    • Primer: RBS-tphB and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphB with RBS 49.2
Miniprep Concentration [ng/µl]
J61002-RBS-tphB 65.8

aroY

  • PCR on pSB1C3-aroY
    • Annealing temperature: 59 °C
    • Primer: RBS-aroY and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
aroY with RBS 55.2
Miniprep Concentration [ng/µl]
J61002-RBS-aroY 77.2


Over expression

tphA1

  • PCR on pSB1C3-tphA1
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphA1 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA1_over-ex 116.2
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA1_over-ex 98.5

tphA2

  • PCR on pSB1C3-tphA2
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphA2 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA2_over-ex 63.9
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA2_over-ex 85.2

tphA3

  • PCR on pSB1C3-tphA3
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphA3 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA3_over-ex 90.4
Miniprep Concentration [ng/µl]
pPR-IBA2-tphA3_over-ex 85.9

tphB

  • PCR on pSB1C3-tphB
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphB and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphB_over-ex 87.5
Miniprep Concentration [ng/µl]
pPR-IBA2-tphB_over-ex 85.2

aroY

  • PCR on pSB1C3-aroY
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-aroY and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
aroY_over-ex 105.1
Miniprep Concentration [ng/µl]
pPR-IBA2-aroY_over-ex 92.1

week 17 (03.-07.09.12)

Operon construction

RBS-tphA1-RBS-tphA2

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

RBS-tphA3-RBS-tphB

  • restriction digest of J61002-RBS-tphA3 by EcoRI and SpeI
  • Purification of insert RBS-tphA3 (cut with EcoRI and SpeI) via gel extraction
Insert Concentration [ng/µl]
RBS-tphA3 (cut with EcoRI and SpeI) 178.9
Miniprep Concentration [ng/µl]
J61002-RBS-tphA3-RBS-tphB 225.5

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

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

Overexpression

tphA2

tphB

aroY

tphA1

tphA3

SDS-PAGE of tphB overexpression and tphA2 overexpression respectively

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

SDS-PAGE of overexpression from all five genes

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

week 18 (10.-17.09.12)

Purification of aroY

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

Purification of TphA3

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

Purification of TphA1

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

week 19 (17.-21.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other

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