Team:TU Darmstadt/Labjournal/Metabolism

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<span style="font-size:200%;"><span style="color:#00689D;">Labjournal Metabolism</span></span>
<span style="font-size:200%;"><span style="color:#00689D;">Labjournal Metabolism</span></span>
 +
This lab journal describes a isolation and characterisation of the terephthalic acid 1,2-dioxigenase system and an dihydrodiol decarboxylase from [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Bacteria Comamonas testosteroni KF-1] in Escherichia Coli. The strain was purchased from DSMZ-German Collection of Microorganism and Cell Cultures (DSMZ no.[http://www.dsmz.de/catalogues/details/culture/DSM-14576.html?tx_dsmzresources_pi5%5BreturnPid%5D=304 14576]). For more informations you will see our [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism project discription].
 +
[[File:Klonierung_sascha.png|900px]]
==week 1 (14.-18.05.12)==
==week 1 (14.-18.05.12)==
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==week 4 (04.-08.06.12)==
==week 4 (04.-08.06.12)==
'''Other'''
'''Other'''
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [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 170: Line 170:
[[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)]]
[[File:WIKI-2012-06-06-_xylE_plasmid_restriction.jpg|thumb|none|alt=A|restriction  of BBa_K316003 using EcoRI and PstI (1kb DNA ladder, NEB)]]
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] digest of BBa_K316003 by XbaI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_K316003 by XbaI and PstI
** Purification of insert xylE-dT via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
** Purification of insert xylE-dT via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 183: Line 183:
'''Other'''
'''Other'''
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] digest of BBa_J23100 by SpeI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_J23100 by SpeI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the restriction   
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the restriction   
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
+
* After overnight incubation of colony 2 in [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media] with ampicilin a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made
==week 6 (18.-22.06.12)==
==week 6 (18.-22.06.12)==
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'''Other'''
'''Other'''
-
* Funktional testing of BBa_J23100-xylE-dT
+
* Functional testing of BBa_J23100-xylE-dT
-
** We inoculated 2 x 50 mL [[LB-medium]]-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
+
** We inoculated 2 x 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
** After incubation we centrifuged the culture at 4600x g for 10 minutes
** After incubation we centrifuged the culture at 4600x g for 10 minutes
-
** We resuspended each pellet with the 1000 µL pipette in 3 mL PBS buffer and added PBS to 120 ml
+
** We resuspended each pellet in 3 mL PBS buffer and added PBS to 120 ml
** We added 2 mL of 0.5 M catechol solution to the first cell suspension  
** We added 2 mL of 0.5 M catechol solution to the first cell suspension  
** We added 2 ml of 0.5 M protocatechuic acid to the second cell suspenion
** We added 2 ml of 0.5 M protocatechuic acid to the second cell suspenion
** We observed in either colony a colour change from colourless to light yellow.
** We observed in either colony a colour change from colourless to light yellow.
-
*** '''Conclusion: XylE accepted protocatechuic acid as substrate. '''
+
*** '''Conclusion: XylE accepted protocatechuic acid as a substrate. '''
-
* Kinetik mesure of XylE with protocatecuic acid as substrate.
+
* Kinetic assay of XylE with protocatechuic acid as a substrate
-
** We inoculated 50 mL [[LB-medium]]-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
+
** We inoculated 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
** After incubation we centrifuged the culture at 4600x g for 10 minutes
** After incubation we centrifuged the culture at 4600x g for 10 minutes
-
** We resuspended each pellet with the 1000 µL pipette in 3 mL PBS buffer and added PBS to 15 ml
+
** We resuspended each pellet in 3 mL PBS buffer and added PBS to 15 ml
-
** After cell disruption we centrifuged the suspension at 9600 rpm for 20 min and decant the supernatant in a fresh tube.
+
** After cell disruption we centrifuged the suspension at 9600 rpm for 20 min and decanted the supernatant in a fresh tube.
-
** For the kinetik we prepared this standard concentrations:  
+
** For the kinetic measurements we prepared the following standard concentrations:  
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
Line 234: Line 234:
|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
| align="center" height="15" | 5
| align="center" height="15" | 5
-
| align="center" | 12,5
+
| align="center" | 12.5
|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
Line 245: Line 245:
|}
|}
-
* We measure the kinetik at wave length= 380 nM for 2 min and calculate the inital speed. From which we created a Michaelis Menten plot.
+
* For the kinetic assay we measured the absorption at 380 nm with an UV/Vis-spectrometer for 2 min and calculated the initial speed. The gained data was used to created a Michaelis Menten plot.
-
[[File:Bild3ddd.png]]
+
[[File:Bild3ddd.png|550px| Michaelis Menten plot of XylE with protocatechuic acid as substrate ]]
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
Line 272: Line 272:
|- style="font-size:11pt" align="center" valign="bottom"
|- style="font-size:11pt" align="center" valign="bottom"
| align="center" height="15" | 12,5
| align="center" height="15" | 12,5
-
| align="center" | 0,285
+
| align="center" | 0.285
|}
|}
Line 298: Line 298:
| Mutated tphA1-prefix/suffix || 62.0
| Mutated tphA1-prefix/suffix || 62.0
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
Line 316: Line 316:
| tphA3-prefix/suffix || 30.5
| tphA3-prefix/suffix || 30.5
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphA3-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA3-prefix/suffix with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA3-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA3-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 330: Line 330:
| pSB1C3-tphA3-prefix/suffix || 79.6
| pSB1C3-tphA3-prefix/suffix || 79.6
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
+
* [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'''
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| tphB_prefix/suffix || 20.3
| tphB_prefix/suffix || 20.3
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
Line 365: Line 365:
| tphB_prefix/suffix || 52.5
| tphB_prefix/suffix || 52.5
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 379: Line 379:
| pSB1C3-tphB-prefix/suffix || 35.8
| pSB1C3-tphB-prefix/suffix || 35.8
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
+
* [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 396: Line 396:
| Mutated tphA1-prefix/suffix || 34.2
| Mutated tphA1-prefix/suffix || 34.2
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
Line 404: Line 404:
[[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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 413: Line 413:
| pSB1C3-tphA1 || 60.5
| pSB1C3-tphA1 || 60.5
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
[[File:WIKI-2012-08-02_pSB1C3-tphA1_test_restriction.jpg|thumb|none|x300px|alt=A|Test restriction digest of psB1C3-tphA1 with EcoRI and PstI (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
[[File:WIKI-2012-08-02_pSB1C3-tphA1_test_restriction.jpg|thumb|none|x300px|alt=A|Test restriction digest of psB1C3-tphA1 with EcoRI and PstI (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
-
* Preparation for [[sequencing]]
+
* Preparation for sequencing
** Sequence was confirmed
** Sequence was confirmed
Line 425: Line 425:
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-kanamycin with one colony of the transformation and incubation
* [[Miniprep]] of the culture
* [[Miniprep]] of the culture
:{| class="wikitable"
:{| class="wikitable"
Line 441: Line 441:
[[File:WIKI-2012-08-07_pSB1C3-tphA2_colony_PCR.jpg|thumb|none|alt=A|Colony PCR on psB1C3-tphA2. From left to right: colony 1-12 (far right: BenchTop 1kb DNA ladder, Promega)]]
[[File:WIKI-2012-08-07_pSB1C3-tphA2_colony_PCR.jpg|thumb|none|alt=A|Colony PCR on psB1C3-tphA2. From left to right: colony 1-12 (far right: BenchTop 1kb DNA ladder, Promega)]]
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and incubation
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 450: Line 450:
| pSB1C3-tphA2-prefix/suffix || 111.1
| pSB1C3-tphA2-prefix/suffix || 111.1
|}
|}
-
* Preparation for [[sequencing]]
+
* Preparation for sequencing
** Sequence was confirmed
** Sequence was confirmed
Line 458: Line 458:
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-kanamycin with one colony of the transformation and incubation
* [[Miniprep]] of the culture
* [[Miniprep]] of the culture
:{| class="wikitable"
:{| class="wikitable"
Line 472: Line 472:
** The PCR was negative
** The PCR was negative
'''Other'''
'''Other'''
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [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 487: Line 487:
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-chloramphenicol with one colony and incubation
* [[Midiprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 496: Line 496:
| pPR-IBA2 || 127
| pPR-IBA2 || 127
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [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 520: Line 520:
| tphA1 with RBS || 33.5
| tphA1 with RBS || 33.5
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphA1 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphA1 with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA1 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA1 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 4 and incubation
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 4 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 546: Line 546:
| tphA2 with RBS || 46.8
| tphA2 with RBS || 46.8
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphA2 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphA2 with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA2 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA2 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 6 and incubation
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 6 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 575: Line 575:
| tphA3 with RBS || 26.5
| tphA3 with RBS || 26.5
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphA3 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphA3 with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA3 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphA3 (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 6 and incubation
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 6 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 601: Line 601:
| tphB with RBS || 49.2
| tphB with RBS || 49.2
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-tphB with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-tphB with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphB (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-tphB (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 1 and incubation
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 627: Line 627:
| aroY with RBS || 55.2
| aroY with RBS || 55.2
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of RBS-aroY with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of RBS-aroY with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-aroY (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of RBS-aroY (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_14_.2813.-17.08.12.29 J61002 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 2 and incubation
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 2 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 657: Line 657:
| tphA1_over-ex || 116.2
| tphA1_over-ex || 116.2
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphA1_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA1_over-ex with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA1_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA1_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 3 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 683: Line 683:
| tphA2_over-ex || 63.9
| tphA2_over-ex || 63.9
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphA2_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA2_over-ex with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA2_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA2_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 709: Line 709:
| tphA3_over-ex || 90.4
| tphA3_over-ex || 90.4
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphA3_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA3_over-ex with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA3_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphA3_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 4 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 735: Line 735:
| tphB_over-ex || 87.5
| tphB_over-ex || 87.5
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of tphB_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphB_over-ex with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphB_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of tphB_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 761: Line 761:
| aroY_over-ex || 105.1
| aroY_over-ex || 105.1
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of aroY_over-ex with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of aroY_over-ex with EcoRI and PstI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of aroY_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of aroY_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
* [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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 3 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 779: Line 779:
===Operon construction===
===Operon construction===
'''RBS-tphA1-RBS-tphA2'''
'''RBS-tphA1-RBS-tphA2'''
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [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 788: Line 788:
| RBS-tphA1 (cut with EcoRI and SpeI)|| 50.2
| RBS-tphA1 (cut with EcoRI and SpeI)|| 50.2
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of J61002-RBS-tphA2 EcoRI and XbaI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of J61002-RBS-tphA2 EcoRI and XbaI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)and RBS-tphA1 (cut with EcoRI and SpeI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the plasmid backbone J61002-RBS-tphA2 (cut with EcoRI and XbaI)and RBS-tphA1 (cut with EcoRI and SpeI)
Line 797: Line 797:
[[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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 4 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 807: Line 807:
|}
|}
'''RBS-tphA3-RBS-tphB'''
'''RBS-tphA3-RBS-tphB'''
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] digest of J61002-RBS-tphA3 by EcoRI and SpeI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of J61002-RBS-tphA3 by EcoRI and SpeI
* Purification of insert RBS-tphA3 (cut with EcoRI and SpeI) via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
* Purification of insert RBS-tphA3 (cut with EcoRI and SpeI) via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
Line 816: Line 816:
| RBS-tphA3 (cut with EcoRI and SpeI)|| 178.9
| RBS-tphA3 (cut with EcoRI and SpeI)|| 178.9
|}
|}
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] of J61002-RBS-tphB EcoRI and XbaI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of J61002-RBS-tphB EcoRI and XbaI
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)and RBS-tphA3 (cut with EcoRI and SpeI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)and RBS-tphA3 (cut with EcoRI and SpeI)
Line 825: Line 825:
[[File:WIKI-2012-09-03_colony_PCR_Operon_A3-B.jpg|thumb|none|alt=A|Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-9 (far left and right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
[[File:WIKI-2012-09-03_colony_PCR_Operon_A3-B.jpg|thumb|none|alt=A|Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-9 (far left and right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)]]
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-ampicillin with colony 1 and incubation
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 1 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 837: Line 837:
'''RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB'''
'''RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB'''
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up 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 [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 849: Line 849:
[[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)]]
[[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)]]
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction] 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
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphA3-RBS-tphB (cut with EcoRI and XbaI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the plasmid backbone J61002-RBS-tphA3-RBS-tphB (cut with EcoRI and XbaI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the plasmid backbone J61002-RBS-tphA3-RBS-tphB EcoRI (cut with EcoRI and XbaI)and RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI)
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#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)
Line 858: Line 858:
[[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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with colony 2 and incubation
* [[Miniprep]] of the culture and a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Glycerine_stock glycerine stock] was made  
* [[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]
Line 871: Line 871:
'''tphA2'''
'''tphA2'''
-
* Inoculate 10 mL of [[LB-medium]]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
+
* Inoculate 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]  
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-ampicillin with pPR-IBA2-tphA1_over-ex and [[incubate]]
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
* Over expression according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_overexpression protocol]
Line 1,058: Line 1,058:
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-
* Note: We don´t perform an SDS-Page from the purification.
+
* Note: We didn't perform an SDS-Page from the purification.
==week 19 (17.-21.09.12)==
==week 19 (17.-21.09.12)==
 +
===Gel permeation chromatography===
 +
 +
* We performed the GPC with the following conditions:
 +
 +
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| width="134" height="15" | Instrument
 +
| width="124" | Pharmacia FPLC System
 +
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| height="15" | Colum
 +
| Superose 6 10/30
 +
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| height="15" | Mobile phase
 +
| PBS pH 7.4
 +
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| height="15" | Detector
 +
| 280 nm
 +
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| height="15" | Flow rate
 +
| 0.5 ml/min
 +
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| height="15" | Progam
 +
| Isocatic
 +
 +
|}
 +
 +
* We injected 50 µl of fraction 3 from each protein (TphA1, TphA2, TphA3, TphB and AroY) for analysis of the molar mass and the oligomerization.
 +
** Results:
 +
[[File:TphA3.JPG|550px|thumb|left|'''GPC analysis of TphA3'''. The Peak of TphA3 has a retention time of 33 minutes. This retention time corresponds to a molar mass of 52 kDa, approximately.]]
 +
[[File:TphB.JPG|550px|thumb|left|'''GPC analysis of TphB'''. The Peak of TphB has a retention time of 32.5 minutes. This retention time corresponds to a molar mass of 64 kDa, approximately. The peak at minute 13 is an undefined contamination.]]
 +
[[File:AroY.JPG|550px|thumb|left|'''GPC analysis of AroY'''. The Peak of AroY has a retention time of 28 minutes. This retention time corresponds to a molar mass of 285.4 kDa, approximately.]]
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
* Note: The GPC analysis of TphA2 and TphA1 respectively didn't work. The peak at 39 minutes is desthiobiotin from the protein purification.
===Operon Construction===
===Operon Construction===
Line 1,075: Line 1,171:
==week 20 (24.-26.09.12)==
==week 20 (24.-26.09.12)==
-
===Enzyme assys===
+
===Enzyme assays===
-
 
+
-
*TpH enzymes
+
-
 
+
-
**For protocol see here
+
-
**We mesure no activity
+
*'''Tph enzymes'''
 +
**For the protocol, [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Enzyme_assays see here]
 +
**We didn't measure the activity.
-
*AroY
+
*'''AroY'''
 +
**For the protocol, [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Enzyme_assays see here]
 +
**Before the addition of XylE we observed a brown solution. The brown color is typical for 1,2-Benzoquinone.
 +
**After the addition of XylE we observed a very high activity.
-
**For protocol see here
+
[[File:AroY_nachweiß.jpg|thumb|none|alt=A|'''Functional test for AroY:'''<br> '''1)''' AroY with 50 mM Protocatechuate after 24 h; <br>'''2)''' after addition of [http://partsregistry.org/Part:BBa_K316003 XylE] and 10 min incubation]]
-
**Bevor addition of XylE we see a brown solution. The brown color is typical for 1,2-Benzoquinone.
+
-
**After the addition of XylE we see a very high activity.
+
-
[[File:AroY_nachweiß.jpg|thumb|none|alt=A|'''Funktional test for AroY:''' '''1)''' AroY with 50 mM Protocatechuate after 24 h; '''2)''' 1) after addition of XylE and 10 min inkubation]]
+

Latest revision as of 02:10, 27 September 2012


Labjournal Metabolism

This lab journal describes a isolation and characterisation of the terephthalic acid 1,2-dioxigenase system and an dihydrodiol decarboxylase from Comamonas testosteroni KF-1 in Escherichia Coli. The strain was purchased from DSMZ-German Collection of Microorganism and Cell Cultures (DSMZ no.[http://www.dsmz.de/catalogues/details/culture/DSM-14576.html?tx_dsmzresources_pi5%5BreturnPid%5D=304 14576]). For more informations you will see our project discription.

Klonierung sascha.png

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
A
tphA1 (left band at 1000 bp) and tphA3 (right band at 500 bp) isolated with colony PCR from C. testosteroni KF-1 genome (far left: 1kb DNA ladder, NEB)

tphB

  • Isolation of the gene from C. testosteroni KF-1 genome using colony-PCR
    • 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
A
tphB (both single bands at 1000 bp) isolated with colony PCR from C. testosteroni KF-1 genome (far left: 1kb DNA ladder, NEB)


Other

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

  • Functional testing of BBa_J23100-xylE-dT
    • We inoculated 2 x 50 mL LB-media-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
    • After incubation we centrifuged the culture at 4600x g for 10 minutes
    • We resuspended each pellet in 3 mL PBS buffer and added PBS to 120 ml
    • We added 2 mL of 0.5 M catechol solution to the first cell suspension
    • We added 2 ml of 0.5 M protocatechuic acid to the second cell suspenion
    • We observed in either colony a colour change from colourless to light yellow.
      • Conclusion: XylE accepted protocatechuic acid as a substrate.
  • Kinetic assay of XylE with protocatechuic acid as a substrate
    • We inoculated 50 mL LB-media-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
    • After incubation we centrifuged the culture at 4600x g for 10 minutes
    • We resuspended each pellet in 3 mL PBS buffer and added PBS to 15 ml
    • After cell disruption we centrifuged the suspension at 9600 rpm for 20 min and decanted the supernatant in a fresh tube.
    • For the kinetic measurements we prepared the following standard concentrations:
Nummer Standard [mM]
1 1
2 2
3 5
4 10
5 12.5
6 15
7 20
  • For the kinetic assay we measured the absorption at 380 nm with an UV/Vis-spectrometer for 2 min and calculated the initial speed. The gained data was used to created a Michaelis Menten plot.

Michaelis Menten plot of XylE with protocatechuic acid as substrate

[Protocatecuatuic acid] mM Velocity units per minute
1 0
2 0
5 0
10 0
12,5 0.285

week 8 (02.-06.07.12)

  • No work progress

week 9 (09.-13.07.12)

Other

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

week 10 (16.-20.07.12)

tphA1

  • PCR on mutated tphA1
    • Annealing temperature: 59 °C
    • Primer: tphA1-Suffix_R and tphA1-l-Prefix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
Mutated tphA1-prefix/suffix 62.0

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 restriction digest of psB1C3-tphA1 with EcoRI and PstI (GeneRuler 100bp Plus DNA Ladder, Fermentas)
  • Preparation for sequencing
    • Sequence was confirmed

week 13 (06.-10.08.12)

tphA2

  • Reconstitution of the tphA2 gene synthesis
  • Transformation of the tphA2 gene synthesis
  • Inoculation of 10 mL LB-media-kanamycin with one colony of the transformation and incubation
  • Miniprep of the culture
Miniprep Concentration [ng/µl]
tphA2 gene synthesis 112.6
A
Colony PCR on psB1C3-tphA2. From left to right: colony 1-12 (far right: BenchTop 1kb DNA ladder, Promega)
Miniprep Concentration [ng/µl]
pSB1C3-tphA2-prefix/suffix 111.1
  • Preparation for sequencing
    • Sequence was confirmed

week 14 (13.-17.08.12)

aroY

  • Reconstitution of the aroY gene synthesis
  • Transformation of the aroY gene synthesis
  • Inoculation of 10 mL LB-media-kanamycin with one colony of the transformation and incubation
  • Miniprep of the culture
Miniprep Concentration [ng/µl]
aroY gene synthesis 63.25

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
A
Colony PCR on J61002-RBS-tphA1 and J61002-RBS-tphA2 (GeneRuler 100bp Plus DNA Ladder, Fermentas)

tphA3

  • PCR on pSB1C3-tphA3
    • Annealing temperature: 59 °C
    • Primer: RBS-tphA3 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA3 with RBS 26.5
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
A
Colony PCR on J61002-RBS-tphA3, J61002-RBS-tphB and J61002-RBS-aroY (GeneRuler 100bp Plus DNA Ladder, Fermentas)

Over expression

tphA1

  • PCR on pSB1C3-tphA1
    • Annealing temperature: 59 °C
    • Primer: EcoRIGFxa-tphA1 and Suffix
  • Both PCR products were purified via gel extraction
  • Concentrations measured by Nanodrop
PCR product Concentration [ng/µl]
tphA1_over-ex 116.2
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
  • Restriction of J61002-RBS-tphB EcoRI and XbaI
  • Dephosphorylation of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)
  • Ligation of the plasmid backbone J61002-RBS-tphB (cut with EcoRI and XbaI)and RBS-tphA3 (cut with EcoRI and SpeI)
  • Transformation of the ligation mix
  • colony-PCR of the transformation for verification
    • The PCR was positive
A
Colony PCR of J61002-RBS-tphA1-RBS-tphA2; from left to right Colony 1-9 (far left and right: Lambda DNA/Eco47I (AvaII) Marker, 13, Fermentas)
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-media-ampicillin with pPR-IBA2-tphA1_over-ex and incubate
  • Over expression according to standard protocol

tphB

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

aroY

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

tphA1

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

tphA3

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

SDS-PAGE of tphB overexpression and tphA2 overexpression respectively

  • SDS-Page according to standard protocol
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 by aroY

Purification of TphB

A
Fractions of the TphB purfication
Band Sample Fraction
1 TphA1 1
2 TphA1 2
3 [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]
4 TphA1 3
5 TphA1 4
6 TphA1 5
7 TphA1 6
7 TphA1 7

Purification of TphA2

  • Note: We didn't perform an SDS-Page from the purification.

week 19 (17.-21.09.12)

Gel permeation chromatography

  • We performed the GPC with the following conditions:
Instrument Pharmacia FPLC System
Colum Superose 6 10/30
Mobile phase PBS pH 7.4
Detector 280 nm
Flow rate 0.5 ml/min
Progam Isocatic
  • We injected 50 µl of fraction 3 from each protein (TphA1, TphA2, TphA3, TphB and AroY) for analysis of the molar mass and the oligomerization.
    • Results:
GPC analysis of TphA3. The Peak of TphA3 has a retention time of 33 minutes. This retention time corresponds to a molar mass of 52 kDa, approximately.
GPC analysis of TphB. The Peak of TphB has a retention time of 32.5 minutes. This retention time corresponds to a molar mass of 64 kDa, approximately. The peak at minute 13 is an undefined contamination.
GPC analysis of AroY. The Peak of AroY has a retention time of 28 minutes. This retention time corresponds to a molar mass of 285.4 kDa, approximately.






























  • Note: The GPC analysis of TphA2 and TphA1 respectively didn't work. The peak at 39 minutes is desthiobiotin from the protein purification.

Operon Construction

A
Test restriction of J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB, J61002-aroY and BBa_K316003 (far left: Lambda DNA/Eco47I (AvaII) Marker, 13)
  • J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB showed unexpected bands although only one was expected after cutting with SpeI and PstI
  • BBa_K316003 showed two bands when cut with PstI and SpeI and PstI respectively where only one was expected
  • This lead us to the assumption that our PstI enzyme was contaminated with EcoRI (note the slightly differences between BBa_K316003 cut with SpeI and PstI and BBa_K316003 cut with XbaI and PstI)
  • Nevertheless also J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB seemed to possess mutation(s) introducing new recognition sites for SpeI and/or PstI
  • As we lacked time to confirm our assumptions by sequencing we decided to cancel further operon construction

week 20 (24.-26.09.12)

Enzyme assays

  • Tph enzymes
    • For the protocol, see here
    • We didn't measure the activity.
  • AroY
    • For the protocol, see here
    • Before the addition of XylE we observed a brown solution. The brown color is typical for 1,2-Benzoquinone.
    • After the addition of XylE we observed a very high activity.
A
Functional test for AroY:
1) AroY with 50 mM Protocatechuate after 24 h;
2) after addition of [http://partsregistry.org/Part:BBa_K316003 XylE] and 10 min incubation