Team:TU Darmstadt/Labjournal/Metabolism

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<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)==
-
'''tphA1'''
 
-
* No work progress
 
-
'''tphA2'''
 
-
* No work progress
 
-
'''tphA3'''
 
-
* No work progress
 
-
'''tphB'''
 
-
* No work progress
 
-
'''aroY'''
 
-
* No work progress
 
'''Other'''
'''Other'''
* Reconstitution of ''C. testosteroni KF-1'' according to DSMZ [http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/engl_Opening.pdf protocol]
* Reconstitution of ''C. testosteroni KF-1'' according to DSMZ [http://www.dsmz.de/fileadmin/Bereiche/Microbiology/Dateien/Kultivierungshinweise/engl_Opening.pdf protocol]
-
* [[Cultivation]] of ''C. testosteroni KF-1'' on agar plates with [[Medium 1]]
+
* [[Cultivation]] of ''C. testosteroni KF-1'' on agar plates with [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
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Production_of_chemically_competent_cells Production of chemically ] competent ''E. coli DH5α'' and ''E. coli BL21(DE3)pLysS'' cells
==week 2 (21.-25.05.12)==
==week 2 (21.-25.05.12)==
'''tphA1'''
'''tphA1'''
-
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [[colony-PCR]]  
+
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR]  
** Annealing temperature: 49 °C
** Annealing temperature: 49 °C
-
** [[Primer]]: tphA1-l-F and tphA1-l-R
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#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]
 +
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
::{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| tphA1 || 0.6
 +
|}
-
'''tphA2'''
 
-
* No work progress
 
'''tphA3'''
'''tphA3'''
-
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [[colony-PCR]]  
+
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR]  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: tphA3-l-F and tphA3-l-R
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#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]
 +
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
::{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| tphA3 || 0.1
 +
|}
 +
 
 +
[[File:WIKI-2012-05-23_colony_PCR_cTestosteroni_A1_A3.png|thumb|none|alt=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'''
'''tphB'''
-
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [[colony-PCR]]  
+
* Isolation of the gene from ''C. testosteroni KF-1'' genome using [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR]  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: tphB-l-F and tphB-l-R
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphB-l-F and tphB-l-R
-
'''aroY'''
+
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* No work progress
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
::{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| tphB || 0.1
 +
|}
 +
 
 +
[[File:WIKI-2012-05-23_colony_PCR_cTestosteroni_B.png|thumb|none|alt=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'''
'''Other'''
-
* [[Transformation]] and [[midi prep]] of all used [[Biobricks]]
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Plasmid_Midiprep midi prep] of all used [[Biobricks]]
-
** Concentrations measured by [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
! Biobrick!! Concentration [ng/µl]
! Biobrick!! Concentration [ng/µl]
|-
|-
-
| BBa_K316003 || -
+
| BBa_K316003 || 114.9
|-
|-
-
| BBa_J23100 || -
+
| BBa_J23100 || 450.2
|-
|-
-
| BBa_B0015 || -
+
| BBa_B0015 || 314.1
|-
|-
-
| BBa_J61101 || -
+
| BBa_J61101 || 86.1
|}
|}
Line 53: Line 115:
* Two PCRs were performed to mutate the PstI site in the wild type gene. The primer tphA1-l-PstI(99)-R and tphA1-l-PstI(99)-F respectively introduced a BsaI site
* Two PCRs were performed to mutate the PstI site in the wild type gene. The primer tphA1-l-PstI(99)-R and tphA1-l-PstI(99)-F respectively introduced a BsaI site
** tphA1 fragment 1
** tphA1 fragment 1
-
** [[PCR]] on tphA1 isolated from ''C. tesosteroni''  
+
** [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/Materials/Metabolism#Primer Primer]: tphA1-l-PstI(99)-R and tphA1-l-R
** tphA1 fragment 2
** tphA1 fragment 2
-
** [[PCR]] on tphA1 isolated from ''C. tesosteroni''  
+
** [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/Materials/Metabolism#Primer Primer]: tphA1-l-PstI(99)-F and tphA1-l-F
-
** Both PCR products were purified via [[gel extraction]]
+
** Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
** Concentrations measured by [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| Fragment 1 || -
+
| Fragment 1 || 40.3
|-
|-
-
| Fragment 2 || -
+
| Fragment 2 || 62.1
|}
|}
-
* Both fragments were cut with BsaI in a [[restriction digest]]
+
[[File:WIKI-2012-05-29_tphA1_mutation.jpg|thumb|none|alt=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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction]
** The ligation mix differed from our standard protocol in the following manner
** The ligation mix differed from our standard protocol in the following manner
*** 100 ng of fragment 1
*** 100 ng of fragment 1
Line 79: Line 144:
*** add DI water up to 20 µL
*** add DI water up to 20 µL
*** incubate for 15 minutes at 37 °C
*** incubate for 15 minutes at 37 °C
-
** [[PCR]] on ligation mix  
+
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on ligation mix  
*** Annealing temperature: 59 °C
*** Annealing temperature: 59 °C
-
*** [[Primer]]: tphA1-l-R and tphA1-l-F
+
*** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-l-R and tphA1-l-F
-
** The PCR product was purified via [[gel extraction]]
+
** The PCR product was purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
** Concentrations measured by [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| Mutated tphA1 || -
+
| Mutated tphA1 || 86.1
|}
|}
-
'''tphA2'''
 
-
* No work progress
 
-
'''tphA3'''
 
-
* No work progress
 
-
'''tphB'''
 
-
* No work progress
 
-
'''aroY'''
 
-
* No work progress
 
-
'''Other'''
 
==week 4 (04.-08.06.12)==
==week 4 (04.-08.06.12)==
-
'''tphA1'''
 
-
 
-
'''tphA2'''
 
-
 
-
'''tphA3'''
 
-
 
-
'''tphB'''
 
-
 
-
'''aroY'''
 
-
 
'''Other'''
'''Other'''
-
* [[Restriction]] digest of BBa_K316003 by EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_K316003 by EcoRI and PstI
-
** Purification of plasmid backbone pSB1C3 via [[gel extraction]]
+
** Purification of plasmid backbone pSB1C3 via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
** Concentrations measured by [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
! Plamid backbone!! Concentration [ng/µl]
! Plamid backbone!! Concentration [ng/µl]
|-
|-
-
| pSB1C3 || -
+
| pSB1C3 || 42.6
|}
|}
-
* [[Restriction]] digest of BBa_K316003 by XbaI and PstI
+
 
-
** Purification of insert xylE-dT via [[gel extraction]]
+
[[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)]]
-
** Concentrations measured by [[Nanoprop]]
+
 
 +
* [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]
 +
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
-
! Plamid backbone!! Concentration [ng/µl]
+
! Insert!! Concentration [ng/µl]
|-
|-
-
| xylE-dT || -
+
| xylE-dT || 22.2
|}
|}
==week 5 (11.-15-06.12)==
==week 5 (11.-15-06.12)==
-
'''tphA1'''
 
-
 
-
'''tphA2'''
 
-
 
-
'''tphA3'''
 
-
 
-
'''tphB'''
 
-
 
-
'''aroY'''
 
'''Other'''
'''Other'''
-
* [[Restriction]] digest of BBa_J23100 by SpeI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_J23100 by SpeI and PstI
-
* [[Dephosphorylation]] of the restriction digest
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Dephosphorylation Dephosphorylation] of the restriction
-
* [[Ligation]] of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of BBa_J23100 (cut with SpeI and PstI) and xylE-dT (cut with XbaI and PstI)
-
** [[Transformation]] with 2 µL of the ligation mix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the ligation mix
-
* [[Colony-PCR]] of the transformation for verification
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] of the transformation for verification
-
* After overnight [[incubation]] of colony x in [[LB medium]] with ampicilin a [[glycerine stock]] was made
+
* After overnight incubation of colony 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)==
Line 154: Line 194:
==week 7 (25.-29.06.12)==
==week 7 (25.-29.06.12)==
-
'''tphA1'''
 
-
'''tphA2'''
+
'''Other'''
 +
* Functional testing of 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
 +
** 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. '''
-
'''tphA3'''
+
* Kinetic assay of XylE with protocatechuic acid as a substrate
 +
** 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
 +
** 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:
 +
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
 +
|- style="font-size:11pt"  valign="bottom"
 +
| width="60" height="15" |''' Nummer'''
 +
| width="120" |''' Standard [mM]'''
-
'''tphB'''
+
|- style="font-size:11pt"  valign="bottom"
 +
| align="center" height="15" | 1
 +
| align="center" | 1
-
'''aroY'''
+
|- style="font-size:11pt"  valign="bottom"
 +
| align="center" height="15" | 2
 +
| align="center" | 2
-
'''Other'''
+
|- style="font-size:11pt"  valign="bottom"
-
* Funktional testing of BBa_J23100-xylE-dT
+
| align="center" height="15" | 3
-
** We inoculated 50 mL [[LB-medium]]-ampicilin with 10 µl of the glycerine stock BBa_J23100-xylE-dT
+
| align="center" | 5
-
** 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)==
+
|- style="font-size:11pt"  valign="bottom"
-
* No work progress
+
| align="center" height="15" | 4
 +
| align="center" | 10
-
==week 9 (09.-13.07.12)==
+
|- style="font-size:11pt"  valign="bottom"
-
'''tphA1'''
+
| align="center" height="15" | 5
 +
| align="center" | 12.5
-
'''tphA2'''
+
|- style="font-size:11pt"  valign="bottom"
 +
| align="center" height="15" | 6
 +
| align="center" | 15
-
'''tphA3'''
+
|- style="font-size:11pt"  valign="bottom"
 +
| align="center" height="15" | 7
 +
| align="center" | 20
-
'''tphB'''
+
|}
 +
* 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.
-
'''aroY'''
+
[[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>
 +
|- style="font-size:11pt;font-weight:bold" align="center" valign="bottom"
 +
| width="134" height="15" | [Protocatecuatuic acid] mM
 +
| width="124" | Velocity units per minute
 +
 
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| align="center" height="15" | 1
 +
| align="center" | 0
 +
 
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| align="center" height="15" | 2
 +
| align="center" | 0
 +
 
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| align="center" height="15" | 5
 +
| align="center" | 0
 +
 
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| align="center" height="15" | 10
 +
| align="center" | 0
 +
 
 +
|- style="font-size:11pt" align="center" valign="bottom"
 +
| align="center" height="15" | 12,5
 +
| align="center" | 0.285
 +
 
 +
|}
 +
 
 +
==week 8 (02.-06.07.12)==
 +
* No work progress
 +
 
 +
==week 9 (09.-13.07.12)==
'''Other'''
'''Other'''
Line 192: Line 287:
==week 10 (16.-20.07.12)==
==week 10 (16.-20.07.12)==
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on mutated tphA1  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on mutated tphA1  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: tphA1-Suffix_R and tphA1-l-Prefix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-Suffix_R and tphA1-l-Prefix
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| Mutated tphA1-prefix/suffix || -
+
| Mutated tphA1-prefix/suffix || 62.0
|}
|}
-
* [[Restriction digest]] of mutated tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
-
* [[Ligation]] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was negative
** The PCR was negative
-
'''tphA2'''
 
'''tphA3'''
'''tphA3'''
-
* [[PCR]] on tphA3 isolated from ''C. testosteroni''  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphA3 isolated from ''C. testosteroni''  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: tphA3-Prefix_F and tphA3-Suffix_R
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA3-Prefix_F and tphA3-Suffix_R
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| tphA3-prefix/suffix || -
+
| tphA3-prefix/suffix || 30.5
|}
|}
-
* [[Restriction digest]] of mutated tphA3-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA3-prefix/suffix with EcoRI and PstI
-
* [[Ligation]] of mutated tphA3-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA3-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-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 [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-tphA3-prefix/suffix || -
+
| pSB1C3-tphA3-prefix/suffix || 79.6
|}
|}
-
* [[Restriction digest]] of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of pSB1C3-tphA3-prefix/suffix with EcoRI and PstI
-
* Preparation for [[sequencing]]
+
* Preparation for sequencing
** Sequence was confirmed  
** Sequence was confirmed  
'''tphB'''
'''tphB'''
-
* [[PCR]] on tphB isolated from ''C. testosteroni''  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphB isolated from ''C. testosteroni''  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: tphB-Prefix and tphB-Suffix_R
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphB-Prefix and tphB-Suffix_R
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| tphB_prefix/suffix || -
+
| tphB_prefix/suffix || 20.3
|}
|}
-
* [[Restriction digest]] of mutated tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
-
* [[Ligation]] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was negative
** The PCR was negative
-
'''aroY'''
 
-
 
-
'''Other'''
 
==week 11 (23.-27.07.12)==
==week 11 (23.-27.07.12)==
-
'''tphA1'''
 
-
 
-
'''tphA2'''
 
-
 
-
'''tphA3'''
 
'''tphB'''
'''tphB'''
-
* [[PCR]] on tphB isolated from ''C. testosteroni''  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on tphB isolated from ''C. testosteroni''  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: tphB-Prefix and tphB-Suffix_R
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphB-Prefix and tphB-Suffix_R
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| tphB_prefix/suffix || -
+
| tphB_prefix/suffix || 52.5
|}
|}
-
* [[Restriction digest]] of mutated tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphB-prefix/suffix with EcoRI and PstI
-
* [[Ligation]] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphB-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
+
* [[Inoculation]] of 10 mL of [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB-media]-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 [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-tphB-prefix/suffix || -
+
| pSB1C3-tphB-prefix/suffix || 35.8
|}
|}
-
* [[Restriction digest]] of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of pSB1C3-tphB-prefix/suffix with EcoRI and PstI
-
* Preparation for [[sequencing]]
+
* Preparation for sequencing
-
** Sequence was confirmed  
+
** Sequence was confirmed
-
'''aroY'''
+
-
 
+
-
'''Other'''
+
==week 12 (30.07.-03.08.12)==
==week 12 (30.07.-03.08.12)==
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on mutated tphA1  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on mutated tphA1  
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: tphA1-Suffix_R and tphA1-l-Prefix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: tphA1-Suffix_R and tphA1-l-Prefix
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| Mutated tphA1-prefix/suffix || -
+
| Mutated tphA1-prefix/suffix || 34.2
|}
|}
-
* [[Restriction digest]] of mutated tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of mutated tphA1-prefix/suffix with EcoRI and PstI
-
* [[Ligation]] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of mutated tphA1-prefix/suffix (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony 1 and [[incubation]]
+
 
-
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
+
[[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)]]
-
* Concentrations measured by [[Nanoprop]]
+
 
 +
* [[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  
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-tphA1 || -
+
| pSB1C3-tphA1 || 60.5
|}
|}
-
* [[Restriction digest]] of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of pSB1C3-tphA1-prefix/suffix with EcoRI and PstI
-
* Preparation for [[sequencing]]
+
-
** Sequence was confirmed
+
-
'''tphA2'''
+
[[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)]]
-
'''tphA3'''
+
* Preparation for sequencing
-
 
+
** Sequence was confirmed
-
'''tphB'''
+
-
 
+
-
'''aroY'''
+
-
 
+
-
'''Other'''
+
==week 13 (06.-10.08.12)==
==week 13 (06.-10.08.12)==
-
'''tphA1'''
 
'''tphA2'''
'''tphA2'''
* Reconstitution of the tphA2 gene synthesis  
* Reconstitution of the tphA2 gene synthesis  
-
* [[Transformation]] of the tphA2 gene synthesis
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the tphA2 gene synthesis
-
* Inoculation of 10 mL [[LB-medium]]-kanamycin with one colony of the transformation and [[incubation]]
+
* Inoculation of 10 mL [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 353: Line 431:
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| tphA2 gene synthesis || -
+
| tphA2 gene synthesis || 112.6
|}
|}
*[[Restriktion digest]] of the tphA2 gene synthesis with EcoRI and PstI
*[[Restriktion digest]] of the tphA2 gene synthesis with EcoRI and PstI
-
*[[Ligation]] of the tphA2 gene synthesis and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+
*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the tphA2 gene synthesis and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol with colony XX and [[incubation]]
+
 
-
* [[Miniprep]] of the culture and a [[glycerine stock]] was made  
+
[[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)]]
-
* Concentrations measured by [[Nanoprop]]
+
 
 +
* [[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  
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-tphA2-prefix/suffix || -
+
| pSB1C3-tphA2-prefix/suffix || 111.1
|}
|}
-
* Preparation for [[sequencing]]
+
* Preparation for sequencing
-
** Sequence was confirmed
+
** Sequence was confirmed
-
 
+
-
'''tphA3'''
+
-
 
+
-
'''tphB'''
+
-
 
+
-
'''aroY'''
+
-
 
+
-
'''Other'''
+
==week 14 (13.-17.08.12)==
==week 14 (13.-17.08.12)==
-
'''tphA1'''
 
-
 
-
'''tphA2'''
 
-
 
-
'''tphA3'''
 
-
 
-
'''tphB'''
 
'''aroY'''
'''aroY'''
* Reconstitution of the aroY gene synthesis  
* Reconstitution of the aroY gene synthesis  
-
* [[Transformation]] of the aroY gene synthesis
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of the aroY gene synthesis
-
* Inoculation of 10 mL [[LB-medium]]-kanamycin with one colony of the transformation and [[incubation]]
+
* Inoculation of 10 mL [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 398: Line 464:
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| tphA2 gene synthesis || -
+
| aroY gene synthesis || 63.25
|}
|}
*[[Restriktion digest]] of the aroY gene synthesis with EcoRI and PstI
*[[Restriktion digest]] of the aroY gene synthesis with EcoRI and PstI
-
*[[Ligation]] of the aroY gene synthesis and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
+
*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Ligation Ligation] of the aroY gene synthesis and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_4_.2804.-08.06.12.29 pSB1C3 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was negative
** The PCR was negative
'''Other'''
'''Other'''
-
* [[Restriction]] digest of BBa_J23100 by EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] digest of BBa_J23100 by EcoRI and PstI
-
** Purification of plasmid backbone J61002 via [[gel extraction]]
+
** Purification of plasmid backbone J61002 via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
** Concentrations measured by [[Nanoprop]]
+
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
::{| class="wikitable"
::{| class="wikitable"
|-
|-
! Plamid backbone!! Concentration [ng/µl]
! Plamid backbone!! Concentration [ng/µl]
|-
|-
-
| J61002 || -
+
| J61002 || 42.5
|}
|}
==week 15 (20.-24.08.12)==
==week 15 (20.-24.08.12)==
-
'''tphA1'''
 
 +
'''Other'''
 +
* 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
 +
* [[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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Midiprep!! Concentration [ng/µl]
 +
|-
 +
| pPR-IBA2 || 127
 +
|}
 +
* [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]
 +
** Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
::{| class="wikitable"
 +
|-
 +
! Plamid backbone!! Concentration [ng/µl]
 +
|-
 +
| pPR-IBA2 || 35.6
 +
|}
 +
 +
==week 16 (27.-31.08.12)==
 +
===Operon construction===
 +
'''tphA1'''
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA1
 +
** Annealing temperature: 59 °C
 +
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#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]
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| 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#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#PCR colony-PCR] for verification of the transformation
 +
** The PCR was positive
 +
* [[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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-tphA1 || 79,6
 +
|}
'''tphA2'''
'''tphA2'''
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA2
 +
** Annealing temperature: 59 °C
 +
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#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]
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| 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#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#PCR colony-PCR] for verification of the transformation
 +
** The PCR was positive
 +
* [[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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-tphA2 || 80.3
 +
|}
-
'''tphA3'''
+
[[File:WIKI-2012-08-29_Colony_PCR_RBS_A1_RBS_A2.png|thumb|none|alt=A|Colony PCR on J61002-RBS-tphA1 and J61002-RBS-tphA2 (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
 +
'''tphA3'''
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA3
 +
** Annealing temperature: 59 °C
 +
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#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]
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| 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#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#PCR colony-PCR] for verification of the transformation
 +
** The PCR was positive
 +
* [[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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-tphA3 || 67.5
 +
|}
'''tphB'''
'''tphB'''
-
 
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphB
 +
** Annealing temperature: 59 °C
 +
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#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]
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| 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#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#PCR colony-PCR] for verification of the transformation
 +
** The PCR was positive
 +
* [[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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-tphB || 65.8
 +
|}
'''aroY'''
'''aroY'''
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-aroY
 +
** Annealing temperature: 59 °C
 +
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#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]
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! PCR product!! Concentration [ng/µl]
 +
|-
 +
| 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#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#PCR colony-PCR] for verification of the transformation
 +
** The PCR was positive
 +
* [[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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-aroY || 77.2
 +
|}
-
'''Other'''
+
[[File:WIKI-2012-08-29_Colony_PCR_RBS_A3_RBS_B_RBS_aroY.png|thumb|none|alt=A|Colony PCR on J61002-RBS-tphA3, J61002-RBS-tphB and J61002-RBS-aroY (GeneRuler 100bp Plus DNA Ladder, Fermentas)]]
-
* Designing primers for over expression and operon construction
+
-
==week 16 (27.-31.08.12)==
+
===Over expression===
-
===Operon construction===
+
'''tphA1'''
'''tphA1'''
-
* [[PCR]] on pSB1C3-tphA1
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA1
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: RBS-tphA1 and Suffix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphA1 and Suffix
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| tphA1 with RBS || -
+
| tphA1_over-ex || 116.2
|}
|}
-
* [[Restriction digest]] of RBS-tphA1 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA1_over-ex with EcoRI and PstI
-
* [[Ligation]] of RBS-tphA1 (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 tphA1_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol 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 [[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 [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-RBS-tphA1 || -
+
| pPR-IBA2-tphA1_over-ex || 98.5
|}
|}
'''tphA2'''
'''tphA2'''
-
* [[PCR]] on pSB1C3-tphA2
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA2
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: RBS-tphA2 and Suffix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphA2 and Suffix
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| tphA2 with RBS || -
+
| tphA2_over-ex || 63.9
|}
|}
-
* [[Restriction digest]] of RBS-tphA2 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA2_over-ex with EcoRI and PstI
-
* [[Ligation]] of RBS-tphA2 (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 tphA2_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol 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 [[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 [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-RBS-tphA2 || -
+
| pPR-IBA2-tphA2_over-ex || 85.2
|}
|}
'''tphA3'''
'''tphA3'''
-
* [[PCR]] on pSB1C3-tphA3
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphA3
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: RBS-tphA3 and Suffix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphA3 and Suffix
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| tphA3 with RBS || -
+
| tphA3_over-ex || 90.4
|}
|}
-
* [[Restriction digest]] of RBS-tphA3 with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphA3_over-ex with EcoRI and PstI
-
* [[Ligation]] of RBS-tphA3 (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 tphA3_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol 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 [[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 [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-RBS-tphA3 || -
+
| pPR-IBA2-tphA3_over-ex || 85.9
|}
|}
'''tphB'''
'''tphB'''
-
* [[PCR]] on pSB1C3-tphB
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-tphB
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: RBS-tphB and Suffix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-tphB and Suffix
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| tphA1 with RBS || -
+
| tphB_over-ex || 87.5
|}
|}
-
* [[Restriction digest]] of RBS-tphB with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of tphB_over-ex with EcoRI and PstI
-
* [[Ligation]] of RBS-tphB (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 tphB_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol 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 [[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 [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-RBS-tphAB || -
+
| pPR-IBA2-tphB_over-ex || 85.2
|}
|}
'''aroY'''
'''aroY'''
-
* [[PCR]] on pSB1C3-aroY
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR_on_a_DNA_template PCR] on pSB1C3-aroY
** Annealing temperature: 59 °C
** Annealing temperature: 59 °C
-
** [[Primer]]: RBS-aroY and Suffix
+
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/Metabolism#Primer Primer]: EcoRIGFxa-aroY and Suffix
-
* Both PCR products were purified via [[gel extraction]]
+
* Both PCR products were purified via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Gel_and_PCR_Clean-Up gel extraction]
-
* Concentrations measured by [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! PCR product!! Concentration [ng/µl]
! PCR product!! Concentration [ng/µl]
|-
|-
-
| aroY with RBS || -
+
| aroY_over-ex || 105.1
|}
|}
-
* [[Restriction digest]] of RBS-aroY with EcoRI and PstI
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest Restriction] of aroY_over-ex with EcoRI and PstI
-
* [[Ligation]] of RBS-aroY (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 aroY_over-ex (cut with EcoRI and PstI) and [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism#week_15_.2820.-24.08.12.29 pPR-IBA2 (cut with EcoRI and PstI)]
-
* [[Transformation]] of ligation mix  
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Heat_shock_transformation_with_E._coli Transformation] of ligation mix  
-
* [[Colony-PCR]] for verification of the transformation
+
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#PCR colony-PCR] for verification of the transformation
** The PCR was positive
** The PCR was positive
-
* [[Inoculation]] of 10 mL of [[LB-medium]]-chloramphenicol 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 [[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 [[Nanoprop]]
+
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
:{| class="wikitable"
:{| class="wikitable"
|-
|-
! Miniprep!! Concentration [ng/µl]
! Miniprep!! Concentration [ng/µl]
|-
|-
-
| pSB1C3-RBS-aroY || -
+
| pPR-IBA2-aroY_over-ex || 92.1
|}
|}
-
 
-
'''Other'''
 
==week 17 (03.-07.09.12)==
==week 17 (03.-07.09.12)==
-
'''tphA1'''
+
===Operon construction===
 +
'''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
 +
* 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]
 +
::{| class="wikitable"
 +
|-
 +
! Insert!! Concentration [ng/µl]
 +
|-
 +
| 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#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#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
 +
** The PCR was positive
-
'''tphA2'''
+
[[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)]]
-
'''tphA3'''
+
* [[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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-tphA1-RBS-tphA2 || 112.5
 +
|}
 +
'''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
 +
* 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]
 +
::{| class="wikitable"
 +
|-
 +
! Insert!! Concentration [ng/µl]
 +
|-
 +
| 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#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#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
 +
** The PCR was positive
 +
 
 +
[[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 [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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-tphA3-RBS-tphB || 225.5
 +
|}
 +
 
 +
'''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
 +
* 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]
 +
::{| class="wikitable"
 +
|-
 +
! Insert!! Concentration [ng/µl]
 +
|-
 +
| RBS-tphA1-RBS-tphA2 (cut with EcoRI and SpeI)|| 129.5
 +
|}
 +
 
 +
[[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#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#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
 +
** The PCR was positive
 +
 
 +
[[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 [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
 +
* Concentrations measured by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Nanodrop Nanodrop]
 +
:{| class="wikitable"
 +
|-
 +
! Miniprep!! Concentration [ng/µl]
 +
|-
 +
| J61002-RBS-tphA1-RBS-tphA2-RBS-tphA3-RBS-tphB || 312.2
 +
|}
 +
 
 +
===Overexpression===
 +
 
 +
'''tphA2'''
 +
* 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]
'''tphB'''
'''tphB'''
 +
* 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]
'''aroY'''
'''aroY'''
 +
* 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]
-
'''Other'''
+
'''tphA1'''
 +
* 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]
 +
 
 +
'''tphA3'''
 +
* 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]
 +
 
 +
'''SDS-PAGE of tphB overexpression and tphA2 overexpression respectively'''
 +
* SDS-Page according to standard [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#SDS-PAGE protocol]
 +
[[File:TphB_tphA2_overex.jpg|thumb|none|alt=A|Results of the overexpression tphA2/tphB]]
 +
{| class="wikitable"
 +
|-
 +
! 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 [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#SDS-PAGE protocol]
 +
[[File: Overex_all.jpg |thumb|none|alt=A|Results of the overexpression aroY/tphA3/tphA1/tphA2/tphB/]]
 +
{| class="wikitable"
 +
|-
 +
! 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)==
==week 18 (10.-17.09.12)==
-
'''tphA1'''
+
'''Purification of aroY'''
 +
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-
'''tphA2'''
+
[[File:Aro_purificate.jpg|thumb|none|alt=A|Fractions of the aroY purfication]]
-
'''tphA3'''
+
{| class="wikitable"
 +
|-
 +
! 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]||-
 +
|}
-
'''tphB'''
+
'''Purification of TphA3'''
 +
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
-
'''aroY'''
+
[[File:Puri_A3.jpg|thumb|none|alt=A|Fractions of the TphA3 purfication]]
-
'''Other'''
+
{| class="wikitable"
 +
|-
 +
! Band!! Sample !! Fraction
 +
|-
 +
| 1 || [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]||
 +
|-
 +
| 2 || TphA3|| Cell suspension
 +
|-
 +
| 3 || TphA3|| Cytoplasm
 +
|-
 +
| 4 || TphA3|| 1
 +
|-
 +
| 5 || TphA3|| 2
 +
|-
 +
| 6 || TphA3|| 3
 +
|-
 +
| 7 || TphA3|| 4
 +
|-
 +
| 8 || TphA3 || 5
 +
|-
 +
| 9 || TphA3|| 6
 +
|-
 +
| 10 || TphA3|| 7
 +
|-
 +
| 11 || [http://www.applichem.com/fileadmin/produktinfo/a4402_de.pdf Protein marker]||-
 +
|}
 +
 
 +
'''Purification of TphA1'''
 +
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
 +
 
 +
[[File:Puri_A1.jpg|thumb|none|alt=A|Fractions of the TphA1 purfication]]
 +
 
 +
{| class="wikitable"
 +
|-
 +
! 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'''
 +
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
 +
 
 +
[[File:2012-09-26_18.07.04.jpg|thumb|none|alt=A|Fractions of the TphB purfication]]
 +
 
 +
{| class="wikitable"
 +
|-
 +
! 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'''
 +
* Protein purification according to standard strep-tag [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Protein_purification purification protocol ]
 +
 
 +
* Note: We didn't perform an SDS-Page from the purification.
==week 19 (17.-21.09.12)==
==week 19 (17.-21.09.12)==
-
'''tphA1'''
+
===Gel permeation chromatography===
-
'''tphA2'''
+
* We performed the GPC with the following conditions:
-
'''tphA3'''
+
{| 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
-
'''tphB'''
+
|- style="font-size:11pt" align="center" valign="bottom"
 +
| height="15" | Colum
 +
| Superose 6 10/30
-
'''aroY'''
+
|- style="font-size:11pt" align="center" valign="bottom"
 +
| height="15" | Mobile phase
 +
| PBS pH 7.4
-
'''Other'''
+
|- 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.]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
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 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
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 +
 
 +
 
 +
 
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 +
 
 +
 
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 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
* 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===
 +
*After several failed cloning attempts a test [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Restriction_digest restriction digest] was performed
 +
 
 +
[[File:WIKI-2012-09-19_testrestriktion_a1-b_(s%2Bp)_aroy_(x%2Bp)_und_(von_links_nach_rechts)_s_p_s%2Bp_x%2Bp.jpg|thumb|none|alt=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, [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Metabolism#Enzyme_assays see here]
 +
**We didn't measure the activity.
 +
 
 +
*'''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.
 +
 
 +
[[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]]

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