Team:TU Darmstadt/Labjournal/Metabolism

From 2012.igem.org

(Difference between revisions)
(week 1 (14.-18.05.12))
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* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA1-l-F and tphA1-l-R
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 67: Line 67:
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA3-l-F and tphA3-l-R
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 80: Line 80:
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphB-l-F and tphB-l-R
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 112: Line 112:
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
*** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA1-l-PstI(99)-R and tphA1-l-R
** tphA1 fragment 2
** tphA1 fragment 2
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
*** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA1-l-PstI(99)-F and tphA1-l-F
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
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** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
*** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA1-l-R and tphA1-l-F
** The PCR product was purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
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** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
==week 6 (18.-22.06.12)==
==week 6 (18.-22.06.12)==
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* Funktional testing of BBa_J23100-xylE-dT
* 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
** 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
+
** 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 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 added 2 mL of 0.5 M catechol solution to the cell suspension  
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA1-Suffix_R and tphA1-l-Prefix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA3-Prefix_F and tphA3-Suffix_R
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphB-Prefix and tphB-Suffix_R
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphB-Prefix and tphB-Suffix_R
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: tphA1-Suffix_R and tphA1-l-Prefix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 326: Line 326:
[[File:WIKI-2012-07-31_pSB1C3-tphA1_colony_PCR.jpg|thumb|none|alt=A|Colony PCR of pSB1C3-tphA1; from left to right: Colony 1-11 (BenchTop 1kb DNA ladder between colony 6 and 7 and on the far right)]]
[[File:WIKI-2012-07-31_pSB1C3-tphA1_colony_PCR.jpg|thumb|none|alt=A|Colony PCR of pSB1C3-tphA1; from left to right: Colony 1-11 (BenchTop 1kb DNA ladder between colony 6 and 7 and on the far right)]]
-
* [[Inoculation]] of 10 mL of [[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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
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* Reconstitution of the tphA2 gene synthesis  
* Reconstitution of the tphA2 gene synthesis  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the tphA2 gene synthesis
* [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
:{| class="wikitable"
:{| class="wikitable"
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
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* Reconstitution of the aroY gene synthesis  
* Reconstitution of the aroY gene synthesis  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the aroY gene synthesis
* [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
:{| class="wikitable"
:{| class="wikitable"
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* Designing primers for over expression and operon construction
* Designing primers for over expression and operon construction
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of pPR-IBA2  
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: RBS-tphA1 and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 444: Line 444:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: RBS-tphA2 and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 470: Line 470:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 482: Line 482:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: RBS-tphA3 and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 496: Line 496:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 508: Line 508:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: RBS-tphB and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 522: Line 522:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 534: Line 534:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: RBS-aroY and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 548: Line 548:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 563: Line 563:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: EcoRIGFxa-tphA1 and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 577: Line 577:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 589: Line 589:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: EcoRIGFxa-tphA2 and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 603: Line 603:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 615: Line 615:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: EcoRIGFxa-tphA3 and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 629: Line 629:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 641: Line 641:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: EcoRIGFxa-tphB and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 655: Line 655:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 667: Line 667:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template 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
+
** [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism/Materials#Primer Primer]: EcoRIGFxa-aroY and Suffix
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 681: Line 681:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 712: Line 712:
[[File:WIKI-2012-09-03_colony_PCR_RBS-tphA1-RBS-tphA2.jpg|thumb|none|alt=A|Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-4 (far right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
[[File:WIKI-2012-09-03_colony_PCR_RBS-tphA1-RBS-tphA2.jpg|thumb|none|alt=A|Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-4 (far right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
-
* [[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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 737: Line 737:
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 770: Line 770:
[[File:WIKI-2012-09-05_colony_PCR_Operon_A1-A2-A3-B.jpg|thumb|none|alt=A|Colony PCR on J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB; Colony 1-7 from left to right (Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
[[File:WIKI-2012-09-05_colony_PCR_Operon_A1-A2-A3-B.jpg|thumb|none|alt=A|Colony PCR on J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB; Colony 1-7 from left to right (Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
-
* [[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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
Line 783: Line 783:
'''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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]

Revision as of 11:02, 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 [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

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

  • 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

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

  • 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
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 bands over TphA1 represent a contamination of aroY

week 19 (17.-21.09.12)

tphA1

tphA2

tphA3

tphB

aroY

Other