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Team:TU Darmstadt/Labjournal/Degradation
From 2012.igem.org
(Difference between revisions)
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WORK IN PROGRESS DO NO INTERFERE!!! | WORK IN PROGRESS DO NO INTERFERE!!! | ||
= Degradation = | = Degradation = | ||
| - | This page features the work carried out by the [https://2012.igem.org/Team:TU_Darmstadt/Project/Degradation degradation team]. The main objectives were the production of a BioBrick containing | + | This page features the work carried out by the [https://2012.igem.org/Team:TU_Darmstadt/Project/Degradation degradation team]. The main objectives were the production of a BioBrick containing ''[https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC Fusarium solani cutinase]'' or '''[https://2012.igem.org/Team:TU_Darmstadt/Materials/Est13 Est13 esterase]''' two enzymes potentially enabling ''E.coli'' of [https://2012.igem.org/Team:TU_Darmstadt/Materials/PET PET] degradation and over-expression stems for activity screening. |
== Activity tests of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] == | == Activity tests of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] == | ||
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{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
| - | ! Component !! test tube 1 !! test tube 2 !! test tube 3 !! test tube 4 !! test tube 5 | + | ! Component !! test tube 1 !! test tube 2 !! test tube 3 !! test tube 4 !! test tube 5 |
|- | |- | ||
| DYT-medium || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL | | DYT-medium || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL | ||
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==== Trouble shooting ==== | ==== Trouble shooting ==== | ||
| - | * evaluation shows a very high sensisty of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 arabinose inducible promoter [BBa_K808000]] even to low concentrations of [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose | + | * evaluation shows a very high sensisty of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 arabinose inducible promoter [BBa_K808000]] even to low concentrations of [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose L-arabinose] (expression starts at around 0.01%) |
* induction of the [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5α] with 1.5% [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose arabinose] ended fatal due to extrem expression of the transmembrane construct [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 RBS-PhoA-His6tag-pNBEst13-Myctag-EstA] | * induction of the [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5α] with 1.5% [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose arabinose] ended fatal due to extrem expression of the transmembrane construct [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 RBS-PhoA-His6tag-pNBEst13-Myctag-EstA] | ||
* starting test expressions with lower [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose L-arabinose] concentrations ranging from 0.05% - 1% | * starting test expressions with lower [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose L-arabinose] concentrations ranging from 0.05% - 1% | ||
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=== CW 24 === | === CW 24 === | ||
==== Thursday, 14.06.2012 ==== | ==== Thursday, 14.06.2012 ==== | ||
| - | * PCR for protein expression of [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] for protein expression of [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
| - | ** | + | ** each [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] is performed in 5 batches à 50 µL. |
| - | ** T<sub>A</sub> = 57°C, t<sub>A</sub> = 35 s, t<sub>E</sub> = 65 s | + | ** Parameter: T<sub>A</sub> = 57°C, t<sub>A</sub> = 35 s, t<sub>E</sub> = 65 s |
| - | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] on [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEST100 pEST100] vector for expression with pEX vector | |
| - | #: gene of interest: [ | + | #: gene of interest: [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] for designed part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] |
| - | #: primers: [ | + | #: primers: [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX_FsC_His_SfiI_up pEX FsC His SfiI up] and [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX_FsC_stop_SfiI_lo pEX FsC Stop SfiI lo] |
==== Friday, 15.06.2012 ==== | ==== Friday, 15.06.2012 ==== | ||
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] for quality control [[File:TU_Darmstadt_logo.png|200px]] |
=== CW 25 === | === CW 25 === | ||
==== Wednesday, 20.06.2012 ==== | ==== Wednesday, 20.06.2012 ==== | ||
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] for preparation [[File:TU_Darmstadt_logo.png|200px]] |
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] products |
| - | *: Concentration of produced [ | + | *: Concentration of produced [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence: 40 ng/µL |
==== Thursday, 21.06.2012 ==== | ==== Thursday, 21.06.2012 ==== | ||
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] product from 14.06. |
| - | ** Enzymes used: [ | + | ** Enzymes used: [https://2012.igem.org/Team:TU_Darmstadt/Materials/SfiI SfiI] |
| - | ** NEBuffer: 4 | + | ** [https://2012.igem.org/Team:TU_Darmstadt/Materials/NEB_buffers#NEBuffer_4_.28green.29 NEBuffer: 4] |
** Digestion time: over night | ** Digestion time: over night | ||
** Digestion temperature: 50°C | ** Digestion temperature: 50°C | ||
==== Friday, 22.06.2012 ==== | ==== Friday, 22.06.2012 ==== | ||
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX] vector |
*: Concentration range: 240-488 ng/µL | *: Concentration range: 240-488 ng/µL | ||
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX] |
| - | ** Enzymes used: [ | + | ** Enzymes used: [https://2012.igem.org/Team:TU_Darmstadt/Materials/SfiI SfiI] |
** NEBuffer: 4 | ** NEBuffer: 4 | ||
** Digestion time: 3 days | ** Digestion time: 3 days | ||
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=== CW 26 === | === CW 26 === | ||
==== Monday, 25.06.2012 ===== | ==== Monday, 25.06.2012 ===== | ||
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] for quality control [[File:TU_Darmstadt_logo.png|200px]] |
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX] [[File:TU_Darmstadt_logo.png|200px]] and digested [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence from 21.06. [[File:TU_Darmstadt_logo.png|200px]] |
| - | *: Concentrations: [ | + | *: Concentrations: [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX]: 77 ng/µL, [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence: 18 ng/µL |
| - | * [[Ligation]] of [ | + | * [[Ligation]] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence with the ratio 1:3 and 1:5 |
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
! Component !! 1:3 !! 1:5 | ! Component !! 1:3 !! 1:5 | ||
|- | |- | ||
| - | | [ | + | | [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence || 1.12 µL || 2.2 µL |
|- | |- | ||
| - | | [ | + | | [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX] || 0.64 µL || 0.64 µL |
|- | |- | ||
| Ligase buffer || 4 µL || 4 µL | | Ligase buffer || 4 µL || 4 µL | ||
|- | |- | ||
| - | | [ | + | | [https://2012.igem.org/Team:TU_Darmstadt/Materials/T4_DNA_Ligase T4 DNA ligase] || 1 µL || 1 µL |
|- | |- | ||
| H<sub>2</sub>O || 33 µL || 30 µL | | H<sub>2</sub>O || 33 µL || 30 µL | ||
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==== Tuesday, 26.06.2012 ==== | ==== Tuesday, 26.06.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation Bacterial transformation] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/TOP10 TOP10] with the ligation product [[pEX-FsC]] from 25.06. |
==== Wednesday, 27.06.2012 ==== | ==== Wednesday, 27.06.2012 ==== | ||
| - | * Two positive clones on [ | + | * Two positive clones on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB] [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM] plates picked for liquid culture |
| - | *: 50 mL [ | + | *: 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM] cultures at 180 rmp and 37°C, over night |
==== Thursday, 28.06.2012 ==== | ==== Thursday, 28.06.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of [[pEX-Fsc]] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/TOP10 TOP10] |
*: Concentration: 45 ng/µL and 405 ng/µL | *: Concentration: 45 ng/µL and 405 ng/µL | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Bacterial_transformation Bacterial transformation] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [[BmH7118]] with the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] product [[pEX-FsC]] |
=== CW 27 === | === CW 27 === | ||
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==== Tuesday, 03.07.2012 ==== | ==== Tuesday, 03.07.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Purification_of_Periplasmatic_Proteins Purification of Periplasmatic Proteins] for all expression temperatures |
==== Wednesday, 04.07.2012 ==== | ==== Wednesday, 04.07.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification Protein purification] without cell disrupter |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page#SDS-PAGE_.28Sch.C3.A4gger.29 SDS-PAGE (Schägger)] analysis of all collected samples |
{| class="wikitable" | {| class="wikitable" | ||
|- | |- | ||
! Expression at 16°C !! Expression at 25°C | ! Expression at 16°C !! Expression at 25°C | ||
|- | |- | ||
| - | | [[ | + | | [[File:TU_Darmstadt_logo.png|200px]] || [[File:TU_Darmstadt_logo.png|200px]] |
|- | |- | ||
! Expression at 30°C !! Expression at 37°C | ! Expression at 30°C !! Expression at 37°C | ||
|- | |- | ||
| - | | [[ | + | | [[File:TU_Darmstadt_logo.png|200px]] || [[File:TU_Darmstadt_logo.png|200px]] |
|} | |} | ||
* The best results were maintained at an expression temperature of 30°C | * The best results were maintained at an expression temperature of 30°C | ||
==== Friday, 06.07.2012 ==== | ==== Friday, 06.07.2012 ==== | ||
| - | * Inoculation of 5 ml [ | + | * Inoculation of 5 ml [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM] with [[BmH7118]] containing [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX#pEX-FsC pEX-FsC] |
* Incubation for 3 days at 20°C | * Incubation for 3 days at 20°C | ||
=== CW 28 === | === CW 28 === | ||
==== Monday, 09.07.2012 ==== | ==== Monday, 09.07.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of [[pEX-FsC]] from 06.07. |
*: Concentration: 470 ng/µL | *: Concentration: 470 ng/µL | ||
* Preparation for sequencing at [[Eurofins]] | * Preparation for sequencing at [[Eurofins]] | ||
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==== Thursday, 12.07.2012 ==== | ==== Thursday, 12.07.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification Protein purification] by standard protocol |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page#SDS-PAGE_.28Sch.C3.A4gger.29 SDS-PAGE (Schägger)] analysis of samples [[File:TU_Darmstadt_logo.png|200px]] |
=== CW 29 === | === CW 29 === | ||
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==== Tuesday, 17.07.2012 ==== | ==== Tuesday, 17.07.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification Protein purification] by standard protocol |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page#SDS-PAGE_.28Sch.C3.A4gger.29 SDS-PAGE (Schägger)] analysis of samples [[File:TU_Darmstadt_logo.png|200px]] |
=== CW 31 === | === CW 31 === | ||
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==== Tuesday, 31.07.2012 ==== | ==== Tuesday, 31.07.2012 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification Protein purification] by standard protocol |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page#SDS-PAGE_.28Sch.C3.A4gger.29 SDS-PAGE (Schägger)] analysis of samples [[File:TU_Darmstadt_logo.png|200px]] |
== SOE PCR == | == SOE PCR == | ||
| - | === Week 1 / | + | === Week 1 / CW 17 === |
==== Tuesday, 24.04.12 ==== | ==== Tuesday, 24.04.12 ==== | ||
* Production of [[electrocompetent cells]] [[DH5alpha]] and [[BL21]] | * Production of [[electrocompetent cells]] [[DH5alpha]] and [[BL21]] | ||
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* setting of [[DYT media]] | * setting of [[DYT media]] | ||
* [[Electroporation]] of [[BL21]] with the following plasmids | * [[Electroporation]] of [[BL21]] with the following plasmids | ||
| - | ** [[pEST100]], carrying a CM-resistance, the genes of [[phoA]], [ | + | ** [[pEST100]], carrying a CM-resistance, the genes of [[phoA]], [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] and [[EstA]] |
** [[pET26b(+)]], carrying a Kan-resistance and the gene of [[pNB-Est13]] | ** [[pET26b(+)]], carrying a Kan-resistance and the gene of [[pNB-Est13]] | ||
** [[pET16]], carrying an Amp-resistance , and is needed for our [[SKV]] | ** [[pET16]], carrying an Amp-resistance , and is needed for our [[SKV]] | ||
** transformed Bl21 cells are incubated over night at 37°C in DYT-media and crossed out on LB-Agar plates | ** transformed Bl21 cells are incubated over night at 37°C in DYT-media and crossed out on LB-Agar plates | ||
==== Wednesday, 25.04.12 ==== | ==== Wednesday, 25.04.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the 3 overnigth [[Bl21]] cultures and [[concentration meassurement via Nanodrop]] |
** [[pEST100]] = 140 ng/µL | ** [[pEST100]] = 140 ng/µL | ||
** [[pET26b(+)]] = 200 ng/µL | ** [[pET26b(+)]] = 200 ng/µL | ||
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#: primers: [[SOE A up]] & [[SOE a1 lo]] | #: primers: [[SOE A up]] & [[SOE a1 lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| - | #: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [ | + | #: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
#: primers: [[SOE A up]] & [[SOE a2 lo]] | #: primers: [[SOE A up]] & [[SOE a2 lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| - | #: gene of interest: [ | + | #: gene of interest: [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[EstA part1]] |
#: primers: [[SOE b2 up]] & [[SOE b2 lo]] | #: primers: [[SOE b2 up]] & [[SOE b2 lo]] | ||
# PCR on [[pET26b(+)]] | # PCR on [[pET26b(+)]] | ||
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#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]] | #: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]] | ||
#: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]] | #: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] (Agorese gele elektrophoresis) for qualitiy control |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | === Friday, 27.04.12 === | + | ==== Friday, 27.04.12 ==== |
* clean up of 1. PCR on [[pEST100]] from [[Thursday, 26.04.12]] via [[Ammonium acetate - Ehtanol DNA precipitation]], solved in 54 µl ddH<sub>2</sub>O | * clean up of 1. PCR on [[pEST100]] from [[Thursday, 26.04.12]] via [[Ammonium acetate - Ehtanol DNA precipitation]], solved in 54 µl ddH<sub>2</sub>O | ||
* restriction of cleaned up 1. PCR and [[pET16b]] with XbaI and NdeI over weekend at 37°C | * restriction of cleaned up 1. PCR and [[pET16b]] with XbaI and NdeI over weekend at 37°C | ||
** Annotation: From now on [[SKV]] will be protocolled in [[SKV]] wiki. | ** Annotation: From now on [[SKV]] will be protocolled in [[SKV]] wiki. | ||
| - | == Week 2 / | + | === Week 2 / CW 18 === |
==== Monday, 30.04.12 ==== | ==== Monday, 30.04.12 ==== | ||
* [[SOE PCR]] | * [[SOE PCR]] | ||
** [[pNB-Est13 part1]] & [[pNB-Est13 part2]], primers: [[SOE b1 up]] & [[SOE b1 lo]] | ** [[pNB-Est13 part1]] & [[pNB-Est13 part2]], primers: [[SOE b1 up]] & [[SOE b1 lo]] | ||
| - | ** [[Promo-LacO-RBS-Phoa]] & [ | + | ** [[Promo-LacO-RBS-Phoa]] & [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC], primers: [[SOE A up]] & [[SOE b2 lo]] |
** Annotation: Every PCR is done in 4 assays à 50 µL, T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E1</sub> = 20s, t<sub>E1</sub> = 35s | ** Annotation: Every PCR is done in 4 assays à 50 µL, T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E1</sub> = 20s, t<sub>E1</sub> = 35s | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of SOE PCR |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | * [[SOE PCR]] of [[Promo-LacO-RBS-Phoa-FsC]] worked, [[pNB-Est13]] did not due to missing clean up via [ | + | * [[SOE PCR]] of [[Promo-LacO-RBS-Phoa-FsC]] worked, [[pNB-Est13]] did not due to missing clean up via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis], precipitation is insufficient, we do it again |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR]s |
# PCR on [[pET26b(+)]] | # PCR on [[pET26b(+)]] | ||
#: gene of interest: [[pNB-Est13 part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[pNB-Est13 part2]] | #: gene of interest: [[pNB-Est13 part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[pNB-Est13 part2]] | ||
| Line 341: | Line 341: | ||
#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]] | #: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]] | ||
#: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]] | #: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
** 1. PCR worked well 2. PCR did not | ** 1. PCR worked well 2. PCR did not | ||
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
==== Wednesday, 02.05.12 ==== | ==== Wednesday, 02.05.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR]s |
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]] | #: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]] | ||
| Line 357: | Line 357: | ||
: T<sub>A</sub> = 57°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s | : T<sub>A</sub> = 57°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s | ||
: every PCR is performed in 3 batches à 50 µL | : every PCR is performed in 3 batches à 50 µL | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
* [[Promega gel extraction]] | * [[Promega gel extraction]] | ||
** c(1.PCR)=6 ng/µL | ** c(1.PCR)=6 ng/µL | ||
| Line 370: | Line 370: | ||
: T<sub>A</sub> = 60°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 35 s | : T<sub>A</sub> = 60°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 35 s | ||
==== Tuesday, 03.05.12 ==== | ==== Tuesday, 03.05.12 ==== | ||
| - | * preparative [ | + | * preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of SOE PCR from yesterday |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
** c[[pNB-Est13]] 7 ng/µL | ** c[[pNB-Est13]] 7 ng/µL | ||
==== Friday, 04.05.12 ==== | ==== Friday, 04.05.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR]s |
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]] | #: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]] | ||
| Line 382: | Line 382: | ||
#: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]] | #: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| - | #: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [ | + | #: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
#: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]] | #: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| Line 389: | Line 389: | ||
** annotation: T<sub>A</sub> = 60°C, t<sub>a</sub> = 25 s, t<sub>E</sub> = 35 s | ** annotation: T<sub>A</sub> = 60°C, t<sub>a</sub> = 25 s, t<sub>E</sub> = 35 s | ||
** every PCR is performed in 3 batches à 50 µL | ** every PCR is performed in 3 batches à 50 µL | ||
| - | * preparative [ | + | * preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | === Week 3 / | + | === Week 3 / CW 19 === |
==== Tuesday 08.05.12 ==== | ==== Tuesday 08.05.12 ==== | ||
* [[SOE PCR]] | * [[SOE PCR]] | ||
| Line 414: | Line 414: | ||
#: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]] | #: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| - | #: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [ | + | #: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
#: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]] | #: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| Line 422: | Line 422: | ||
** every PCR is performed in 2 batches à 50 µL | ** every PCR is performed in 2 batches à 50 µL | ||
==== Friday, 09.05.12 ==== | ==== Friday, 09.05.12 ==== | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | * [[ | + | * [[File:TU_Darmstadt_logo.png|200px]] |
* both [[EstA part 1]] (3.PCR & 4.PCR) did not work well | * both [[EstA part 1]] (3.PCR & 4.PCR) did not work well | ||
| - | === Week 4 / | + | === Week 4 / CW 20 === |
==== Monday, 14.05.12 ==== | ==== Monday, 14.05.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of remaining PCRs from [[Wednesday, 09.05.12]] |
** c([[pNB-Est13 part1]])=24 ng/µL | ** c([[pNB-Est13 part1]])=24 ng/µL | ||
** c([[pNB-Est13 part2]])=26 ng/µL | ** c([[pNB-Est13 part2]])=26 ng/µL | ||
| Line 438: | Line 438: | ||
** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 35 s | ** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 35 s | ||
** T<sub>A2</sub> = 57°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 1.15 min | ** T<sub>A2</sub> = 57°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 1.15 min | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
==== Tuesday, 15.05.12 ==== | ==== Tuesday, 15.05.12 ==== | ||
* [[Gel extraction]9 of [[SOE PCR]] from yesterday | * [[Gel extraction]9 of [[SOE PCR]] from yesterday | ||
| Line 451: | Line 451: | ||
#: gene of interest: [[Promo-LacO-RBS-Phoa-FsC]] for [[SOE PCR]] with [[EstA]] | #: gene of interest: [[Promo-LacO-RBS-Phoa-FsC]] for [[SOE PCR]] with [[EstA]] | ||
#: primers: [[SOE A up]] & [[SOE b2 lo]] | #: primers: [[SOE A up]] & [[SOE b2 lo]] | ||
| - | #: template: [ | + | #: template: [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] from [[Thursday, 26.04.12]] & [[Promo-LacO-RBS-Phoa]] from [[Wednesday, 02.05.12]] |
** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 35 s | ** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 35 s | ||
** T<sub>A2</sub> = 57°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 1.15 min | ** T<sub>A2</sub> = 57°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 1.15 min | ||
| Line 457: | Line 457: | ||
==== Wednesday, 16.05.12 ==== | ==== Wednesday, 16.05.12 ==== | ||
* [[SOE PCR]] of [[Promo-LacO-RBS-Phoa-pNBEst13] did not work but [[Promo-LacO-RBS-Phoa-FsC]] worked | * [[SOE PCR]] of [[Promo-LacO-RBS-Phoa-pNBEst13] did not work but [[Promo-LacO-RBS-Phoa-FsC]] worked | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
** c([[Promo-LacO-RBS-Phoa-FsC]])=10 ng/µL | ** c([[Promo-LacO-RBS-Phoa-FsC]])=10 ng/µL | ||
=== Week 5 / Kw 21 === | === Week 5 / Kw 21 === | ||
==== Monday, 23.05.12 ==== | ==== Monday, 23.05.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] |
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]] and [[EstA part2]] | #: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]] and [[EstA part2]] | ||
#: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]] | #: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| - | #: gene of interest: [[EstA part1]] for [[SOE PCR]] with [ | + | #: gene of interest: [[EstA part1]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] and [[EstA part2]] |
#: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]] | #: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]] | ||
==== Tuesday, 22.05.12 ==== | ==== Tuesday, 22.05.12 ==== | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
| - | ** c([[EstA part1]] for [[SOE PCR]] with [ | + | ** c([[EstA part1]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])=4 ng/µL |
** c([[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]])=7 ng/µL | ** c([[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]])=7 ng/µL | ||
==== Wednesday, 23.05.12 ==== | ==== Wednesday, 23.05.12 ==== | ||
| Line 481: | Line 481: | ||
#: template: [[EstA part1]] from yesterday & [[EstA part2]] from [[Monday, 14.05.12]] | #: template: [[EstA part1]] from yesterday & [[EstA part2]] from [[Monday, 14.05.12]] | ||
# [[SOE PCR]] | # [[SOE PCR]] | ||
| - | #: gene of interest: [[EstA]] for [[SOE PCR]] with [ | + | #: gene of interest: [[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
#: primers: [[SOE c2 up]] & [[SOE D lo]] | #: primers: [[SOE c2 up]] & [[SOE D lo]] | ||
#: template: [[EstA part1]] from yesterday & [[EstA part2]] from [[Monday, 14.05.12]] | #: template: [[EstA part1]] from yesterday & [[EstA part2]] from [[Monday, 14.05.12]] | ||
** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> =45 s | ** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> =45 s | ||
** T<sub>A2</sub> = 65°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 90 s | ** T<sub>A2</sub> = 65°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 90 s | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=42 ng/µL | ** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=42 ng/µL | ||
| - | ** c([[EstA]] for [[SOE PCR]] with [ | + | ** c([[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])=23 ng/µL |
==== Thursday, 24.05.12 ==== | ==== Thursday, 24.05.12 ==== | ||
* [[PCR 1]] | * [[PCR 1]] | ||
| Line 496: | Line 496: | ||
#: gene of interest: [[EstA]] for [[SOE PCR]] with [[pNB-Est13]] | #: gene of interest: [[EstA]] for [[SOE PCR]] with [[pNB-Est13]] | ||
#: primers: [[SOE c1 up]] & [[SOE D lo]] | #: primers: [[SOE c1 up]] & [[SOE D lo]] | ||
| - | # PCR on [[EstA]] for [[SOE PCR]] with [ | + | # PCR on [[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
| - | #: gene of interest: [[EstA]] for [[SOE PCR]] with [ | + | #: gene of interest: [[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
#: primers: [[SOE c2 up]] & [[SOE D lo]] | #: primers: [[SOE c2 up]] & [[SOE D lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| Line 503: | Line 503: | ||
#: primers: [[SOE A up]] & [[SOE a1 lo]] | #: primers: [[SOE A up]] & [[SOE a1 lo]] | ||
# PCR on [[pEST100]] | # PCR on [[pEST100]] | ||
| - | #: gene of interest: [ | + | #: gene of interest: [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[EstA part1]] |
#: primers: [[SOE b2 up]] & [[SOE b2 lo]] | #: primers: [[SOE b2 up]] & [[SOE b2 lo]] | ||
** annotation: T<sub>A</sub> = 60°C, t<sub>a</sub> = 25 s, t<sub>E</sub> =45 s | ** annotation: T<sub>A</sub> = 60°C, t<sub>a</sub> = 25 s, t<sub>E</sub> =45 s | ||
: each PCR is performed in 3 batches à 50 µL | : each PCR is performed in 3 batches à 50 µL | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
** both EstAs worked | ** both EstAs worked | ||
** Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13 worked | ** Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13 worked | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=39 ng/µL | ** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=39 ng/µL | ||
| - | ** c([[EstA]] for [[SOE PCR]] with [ | + | ** c([[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])=20 ng/µL |
** c([[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]])=13 ng/µL | ** c([[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]])=13 ng/µL | ||
=== Week 6 / Kw 22 === | === Week 6 / Kw 22 === | ||
| Line 533: | Line 533: | ||
** T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> =45 s | ** T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> =45 s | ||
** T<sub>A2</sub> = 65°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 90 s | ** T<sub>A2</sub> = 65°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 90 s | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
** [[Promo-LacO-RBS-Phoa-Fsc-EstA]] worked | ** [[Promo-LacO-RBS-Phoa-Fsc-EstA]] worked | ||
** [[Promo-LacO-RBS-Phoa-pNBEst13]] worked | ** [[Promo-LacO-RBS-Phoa-pNBEst13]] worked | ||
==== Thursday, 29.05.12 ==== | ==== Thursday, 29.05.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
** c([[Promo-LacO-RBS-Phoa-Fsc-EstA]])= 8 ng/µL | ** c([[Promo-LacO-RBS-Phoa-Fsc-EstA]])= 8 ng/µL | ||
** c([[Promo-LacO-RBS-Phoa-pNBEst13]])= 13 ng/µL | ** c([[Promo-LacO-RBS-Phoa-pNBEst13]])= 13 ng/µL | ||
== SKV == | == SKV == | ||
| - | === Week 1 / | + | === Week 1 / CW 17 === |
==== Tuesday, 24.04.12 ==== | ==== Tuesday, 24.04.12 ==== | ||
* Production of [[electrocompetent cells]] [[DH5alpha]] and [[BL21]] | * Production of [[electrocompetent cells]] [[DH5alpha]] and [[BL21]] | ||
| Line 548: | Line 548: | ||
* setting of [[DYT media]] | * setting of [[DYT media]] | ||
* [[Electroporation]] of [[BL21]] with the following plasmids | * [[Electroporation]] of [[BL21]] with the following plasmids | ||
| - | ** [[pEST100]], carrying a CM-resistance, the genes of [[phoA]], [ | + | ** [[pEST100]], carrying a CM-resistance, the genes of [[phoA]], [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] and [[EstA]] |
** [[pET26b(+)]], carrying a Kan-resistance and the gene of [[pNB-Est13]] | ** [[pET26b(+)]], carrying a Kan-resistance and the gene of [[pNB-Est13]] | ||
** [[pET16]], carrying an Amp-resistance , and is needed for our [[SKV]] | ** [[pET16]], carrying an Amp-resistance , and is needed for our [[SKV]] | ||
==== Wednesday, 25.04.12 ==== | ==== Wednesday, 25.04.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the 3 overnigth [[Bl21]] cultures and [[concentration meassurement via Nanodrop]] |
** [[pEST100]] = 140 ng/µL | ** [[pEST100]] = 140 ng/µL | ||
** [[pET26b(+)]] = 200 ng/µL | ** [[pET26b(+)]] = 200 ng/µL | ||
| Line 562: | Line 562: | ||
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SKV]] | #: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SKV]] | ||
#: primers: [[SKV a1 up XbaI]] & [[SKV a1 lo NdeI]] | #: primers: [[SKV a1 up XbaI]] & [[SKV a1 lo NdeI]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] (Agarose gele elektrophoresis) for qualitiy control |
[[120426 PCR 1-3 siehe Laborbuch 26.4.tif]] | [[120426 PCR 1-3 siehe Laborbuch 26.4.tif]] | ||
| - | === Friday, 27.04.12 === | + | ==== Friday, 27.04.12 ==== |
* clean up of 1. PCR on [[pEST100]] from [[Thursday, 26.04.12]] via [[Ammonium acetate - Ehtanol DNA precipitation]], solved in 54 µl ddH<sub>2</sub>O | * clean up of 1. PCR on [[pEST100]] from [[Thursday, 26.04.12]] via [[Ammonium acetate - Ehtanol DNA precipitation]], solved in 54 µl ddH<sub>2</sub>O | ||
* restriction of cleaned up 1. PCR and [[pET16b]] with XbaI and NdeI over weekend at 37°C | * restriction of cleaned up 1. PCR and [[pET16b]] with XbaI and NdeI over weekend at 37°C | ||
| - | == Week 2 / | + | === Week 2 / CW 18 === |
| - | === Monday, 30.04.12 === | + | ==== Monday, 30.04.12 ==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of DNA digestion from [[Friday, 27.04.12]] |
[[120430 Testrestrikt.XbaINdeI.tif]] | [[120430 Testrestrikt.XbaINdeI.tif]] | ||
| - | * Only one single band on [ | + | * Only one single band on [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] image |
** For saveness two single digests are performed at 20 µL batches. One with XbaI and the other with NdeI. Incubation for 2 hours. | ** For saveness two single digests are performed at 20 µL batches. One with XbaI and the other with NdeI. Incubation for 2 hours. | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of single digests |
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
** Enzymes cut once each, so digest should have worked. | ** Enzymes cut once each, so digest should have worked. | ||
* [[Ligation]] of [[PhoA]] in cut (XbaI / NdeI) [[pET16b(+)]] | * [[Ligation]] of [[PhoA]] in cut (XbaI / NdeI) [[pET16b(+)]] | ||
** Annotation: 30 µL batch, 1:5 ratio, Ligation at 4°C for 2 days till [[Wednesday, 02.05.12]] | ** Annotation: 30 µL batch, 1:5 ratio, Ligation at 4°C for 2 days till [[Wednesday, 02.05.12]] | ||
| - | == | + | === Week 4/CW 20 === |
| - | === Monday, 07.05.=== | + | ==== Monday, 07.05.==== |
*[[Electroporation]] of [[DH5alpha]] with 5 µl of the ligation which was performed 2 days before. | *[[Electroporation]] of [[DH5alpha]] with 5 µl of the ligation which was performed 2 days before. | ||
* clean up of the PCR-product PhoA [[Friday, 04.05.]] | * clean up of the PCR-product PhoA [[Friday, 04.05.]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of half of the product with XbaI/NdeI for 1,5h at 37°C |
| - | * PhoA is cleaned up by [ | + | * PhoA is cleaned up by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
* concentration:7,36 ng/µl,10,6 ng/µl | * concentration:7,36 ng/µl,10,6 ng/µl | ||
*[[Ligation]] of PhoA x [[pET16b(+)]] for 2h, at the rate of (Vector/Insert) 1:5, 1:3 | *[[Ligation]] of PhoA x [[pET16b(+)]] for 2h, at the rate of (Vector/Insert) 1:5, 1:3 | ||
Annotation: If it does not say anything else, [[Ligation]] is always done in 20µl batches. | Annotation: If it does not say anything else, [[Ligation]] is always done in 20µl batches. | ||
| - | === Tuesday, 08.05. === | + | ==== Tuesday, 08.05. ==== |
* Transformation of [[DH5alpha]] with [[PhoA]] x [[pET16b(+)]] was not successfull, so it was performed again [[Monday, 07.05.]] | * Transformation of [[DH5alpha]] with [[PhoA]] x [[pET16b(+)]] was not successfull, so it was performed again [[Monday, 07.05.]] | ||
* DNA digestion of [[pET16b(+)]] | * DNA digestion of [[pET16b(+)]] | ||
| Line 593: | Line 593: | ||
** product is cleaned up by [[Ammonium acetate - Ehtanol DNA precipitation]] | ** product is cleaned up by [[Ammonium acetate - Ehtanol DNA precipitation]] | ||
**concentration: 85 ng/µl | **concentration: 85 ng/µl | ||
| - | === Wednesday, 09.05.=== | + | ==== Wednesday, 09.05.==== |
* [[Colony-PCR]] | * [[Colony-PCR]] | ||
** gene of interest: [[PhoA]] | ** gene of interest: [[PhoA]] | ||
** primer:[[SKV a1 up XbaI]], [[SKV a1 lo NdeI]] | ** primer:[[SKV a1 up XbaI]], [[SKV a1 lo NdeI]] | ||
| - | * PhoA was not inserted, but 2 colonies were picked and inoculated in 5 ml [ | + | * PhoA was not inserted, but 2 colonies were picked and inoculated in 5 ml [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB LB] Amp medium to look at it again due to a test-restriction. |
| - | === Thursday, 10.05.=== | + | ==== Thursday, 10.05.==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of [[PhoA]] x [[pET16b(+)]] |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep]: [[PhoA]] x [[pET16b(+)]] |
** enzymes: XbaI/NdeI, incubation: 1,5 h, 37°C | ** enzymes: XbaI/NdeI, incubation: 1,5 h, 37°C | ||
** digest shows that [[PhoA]] was not inserted | ** digest shows that [[PhoA]] was not inserted | ||
| Line 613: | Line 613: | ||
** [[SKV b2 up NdeI]], [[SKV b2 lo NCOI]] | ** [[SKV b2 up NdeI]], [[SKV b2 lo NCOI]] | ||
** [[SKV c1 up NcoI]], [[SKV c1 lo EcoRI]] | ** [[SKV c1 up NcoI]], [[SKV c1 lo EcoRI]] | ||
| - | === Friday, 11.05. === | + | ==== Friday, 11.05. ==== |
* an analytic, 1% agarose-gel proves that the amplification of [[FSC]], [[Est13]] and [[EstA]] was successfull. | * an analytic, 1% agarose-gel proves that the amplification of [[FSC]], [[Est13]] and [[EstA]] was successfull. | ||
[[Image]] | [[Image]] | ||
| Line 624: | Line 624: | ||
** PhoA= 10,6 ng/µl [[Monday,07.05.]] | ** PhoA= 10,6 ng/µl [[Monday,07.05.]] | ||
** incubation: 2 days, 4°C | ** incubation: 2 days, 4°C | ||
| - | == | + | === Week5/CW21 === |
| - | === Monday, 14.05.=== | + | ==== Monday, 14.05.==== |
| - | * the [ | + | * the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [[pET16b(+)]] cut with XbaI and NdeI is isolated by [[Ammonium acetate - Ehtanol DNA precipitation]] |
* [[LIgation]] of [[phoA]] x [[pET16b(+)]] is used to transform [[DH5alpha]] via [[Electroporation]] | * [[LIgation]] of [[phoA]] x [[pET16b(+)]] is used to transform [[DH5alpha]] via [[Electroporation]] | ||
| - | === Tuesday, 15.05.=== | + | ==== Tuesday, 15.05.==== |
*[[Colony-PCR]] | *[[Colony-PCR]] | ||
**primer: | **primer: | ||
**[[SKV a1 up XbaI]], | **[[SKV a1 up XbaI]], | ||
**[[SKV a1 lo NdeI]] | **[[SKV a1 lo NdeI]] | ||
| - | *[[ | + | *[[File:TU_Darmstadt_logo.png|200px]] |
*PhoA is inserted, so 10 clones are picked to grow them in 5ml DYT over night. | *PhoA is inserted, so 10 clones are picked to grow them in 5ml DYT over night. | ||
| - | === Wednesday, 16.05.=== | + | ==== Wednesday, 16.05.==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of 10 clones, the plasmid-DNA is isolated out of 3ml culture per clone. |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] with NdeI and NcoI of the plasmids [[PhoA]] x [[pET16b(+)]] 1-4, so [[FSC]] and [[Est13]] can be added to the construct. |
* [[FSC]] is cut with NdeI and NcoI | * [[FSC]] is cut with NdeI and NcoI | ||
| - | ** digests are performed in 20µl total volume at 37°C for 1,5h and cleaned up by [ | + | ** digests are performed in 20µl total volume at 37°C for 1,5h and cleaned up by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | * [[ | + | * [[File:TU_Darmstadt_logo.png|200px]] |
* [[PCR1]] of [[EST13]] | * [[PCR1]] of [[EST13]] | ||
** template: product of SOE-PCR, cleaned up by the Promega-Kit | ** template: product of SOE-PCR, cleaned up by the Promega-Kit | ||
** primer: [[SKV b1 up NdeI]], [[SKV b1 lo NcoI]] | ** primer: [[SKV b1 up NdeI]], [[SKV b1 lo NcoI]] | ||
| - | === Friday, 18.05.=== | + | ==== Friday, 18.05.==== |
*[[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]]) | *[[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]]) | ||
** ratio vector/insert: 3:1, 1:1, 1:3 | ** ratio vector/insert: 3:1, 1:1, 1:3 | ||
| Line 650: | Line 650: | ||
*([[phoA]] x [[pET16b(+)]])= 3,72 ng/µl | *([[phoA]] x [[pET16b(+)]])= 3,72 ng/µl | ||
*[[FSC]]= 12,22 ng/µl | *[[FSC]]= 12,22 ng/µl | ||
| - | == | + | === Week 6/CW 22 === |
| - | === Monday, 21.05.=== | + | ==== Monday, 21.05.==== |
* [[Elektroporation]] of [[DH5alpha]] with the [[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]]) | * [[Elektroporation]] of [[DH5alpha]] with the [[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]]) | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of ([[phoA]] x [[pET16b(+)]])(150µl,160µl) for the insertion of [[Est13]]/[[FSC]] and [[Est13]](50µl) |
** enzymes: NdeI, NcoI | ** enzymes: NdeI, NcoI | ||
** incubation: 1,5h, 37°C | ** incubation: 1,5h, 37°C | ||
| - | ** cleaned up via[ | + | ** cleaned up via[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
** c([[phoA]] x [[pET16b(+)]])= 23,5 ng/µl | ** c([[phoA]] x [[pET16b(+)]])= 23,5 ng/µl | ||
** c([[Est13]]= 7,7 ng/µ | ** c([[Est13]]= 7,7 ng/µ | ||
| - | === Tuesday, 22.05.=== | + | ==== Tuesday, 22.05.==== |
* [[Ligation]] of [[Est13]] and ([[phoA]] x [[pET16b(+)]]) | * [[Ligation]] of [[Est13]] and ([[phoA]] x [[pET16b(+)]]) | ||
** both components have been cut with NdeI and NcoI [[Monday, 21.05.]] | ** both components have been cut with NdeI and NcoI [[Monday, 21.05.]] | ||
| Line 665: | Line 665: | ||
** incubation: 2h, 25°C | ** incubation: 2h, 25°C | ||
** [[Electroporation]] of dH5alpha using the [[Ligation]] of [[Est13]] x ([[phoA]] x [[pET16b(+)]], 5 µl per 100µl cells | ** [[Electroporation]] of dH5alpha using the [[Ligation]] of [[Est13]] x ([[phoA]] x [[pET16b(+)]], 5 µl per 100µl cells | ||
| - | === Wednesday, 23.05. === | + | ==== Wednesday, 23.05. ==== |
* [[colony-PCR]] | * [[colony-PCR]] | ||
** aim is to check the insertion of [[Est13]] | ** aim is to check the insertion of [[Est13]] | ||
** Primer:[[SKV b1 up NdeI]], [[SKV b1 lo NcoI]] | ** Primer:[[SKV b1 up NdeI]], [[SKV b1 lo NcoI]] | ||
| - | ** 8 colonies have been picked and the [ | + | ** 8 colonies have been picked and the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] proves, that [[Est 13]] has not been inserted into ([[phoA]] x [[pET16b(+)]] |
| - | === Wednesday, 23.05.=== | + | ==== Wednesday, 23.05.==== |
* [[Ligation]] of [[FSC]] and [[Est13]] in ([[phoA]] x [[pET16b(+)]] | * [[Ligation]] of [[FSC]] and [[Est13]] in ([[phoA]] x [[pET16b(+)]] | ||
** ratio vector/insert: 1:3, 1:5 | ** ratio vector/insert: 1:3, 1:5 | ||
** c(templates):[[Monday, 21.05.]] | ** c(templates):[[Monday, 21.05.]] | ||
*[[Elektroporation]] of [[DH5alpha]] with [[Est13]] in ([[phoA]] x [[pET16b(+)]] | *[[Elektroporation]] of [[DH5alpha]] with [[Est13]] in ([[phoA]] x [[pET16b(+)]] | ||
| - | === Thursday, 24.05. === | + | ==== Thursday, 24.05. ==== |
* [[colony-PCR]] proves, that [[Est13]] has been inserted into ([[phoA]] x [[pET16b(+)]] | * [[colony-PCR]] proves, that [[Est13]] has been inserted into ([[phoA]] x [[pET16b(+)]] | ||
** plates are covered with colonies, 8 colonies are chosen for [[colony-PCR]] | ** plates are covered with colonies, 8 colonies are chosen for [[colony-PCR]] | ||
** colony No.5 is chosen to be inoculated in 5µ DYT/Amp medium | ** colony No.5 is chosen to be inoculated in 5µ DYT/Amp medium | ||
* [PCR 1] is performed again to amplify [[FSC]] and [[Est13]] | * [PCR 1] is performed again to amplify [[FSC]] and [[Est13]] | ||
| - | **products are cleaned up by [ | + | **products are cleaned up by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | **[[ | + | **[[File:TU_Darmstadt_logo.png|200px]] |
| - | === Friday, 25.05. === | + | ==== Friday, 25.05. ==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [[FSC]] and [[Est13]] [[Thursday, 24.05.]] |
** enzymes: NdeI, NcoI | ** enzymes: NdeI, NcoI | ||
** incubation: 2days, 37°C | ** incubation: 2days, 37°C | ||
| - | == week 7/ | + | === week 7/CW 23 === |
| - | === Tuesday, 29.05. === | + | ==== Tuesday, 29.05. ==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [[FSC]] and [[Est13]] is cleaned up via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
* [[MIniprep]] of [[Est13]] x ([[phoA]] x [[pET16b(+)]] via Promega-Kit | * [[MIniprep]] of [[Est13]] x ([[phoA]] x [[pET16b(+)]] via Promega-Kit | ||
* [[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]]) | * [[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]]) | ||
| Line 697: | Line 697: | ||
** 4 assays à 50 µL, T<sub>A</sub> = 66°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90s | ** 4 assays à 50 µL, T<sub>A</sub> = 66°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90s | ||
** cleaned up via Kit | ** cleaned up via Kit | ||
| - | === Wednesday, 30.05. === | + | ==== Wednesday, 30.05. ==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [[EstA]] (product of PCR, 80 µl) and Plasmid: ([[Est13]] x [[phoA]] x [[pET16b(+)]]) |
** enzymes: EcoRI, NcoI | ** enzymes: EcoRI, NcoI | ||
** incubation: 1,5h, 37°C | ** incubation: 1,5h, 37°C | ||
| - | ** [ | + | ** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
** [[Image]] | ** [[Image]] | ||
** c([[EstA]]) = 6,7 ng/µl | ** c([[EstA]]) = 6,7 ng/µl | ||
** c([[Est13]] x [[phoA]] x [[pET16b(+)]])= 23 ng/µl | ** c([[Est13]] x [[phoA]] x [[pET16b(+)]])= 23 ng/µl | ||
* [[Electroporation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]] worked well, plates are covered with colonies which are picked to be grown in 5ml DYT/Amp medium over night. | * [[Electroporation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]] worked well, plates are covered with colonies which are picked to be grown in 5ml DYT/Amp medium over night. | ||
| - | === Thursday, 31.05. === | + | ==== Thursday, 31.05. ==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the amplified ([[FSC]] x [[phoA]] x [[pET16b(+)]]) |
| - | ** [ | + | ** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] to ligate [[EstA]] |
** enzymes:NcoI, EcoRI | ** enzymes:NcoI, EcoRI | ||
** incubation: 1,5h, 37°C | ** incubation: 1,5h, 37°C | ||
| Line 721: | Line 721: | ||
*conditions: | *conditions: | ||
** T<sub>A</sub> = 66°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90s | ** T<sub>A</sub> = 66°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90s | ||
| - | * the [ | + | * the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] shows, that there is no binding-specifity concerning the genes of interest and the primers, but primers have been checked on mistakes. |
* to solve the problem, the [[LIgation]] of [[FSC]] and [[Est13]] shall be repeated from the beginning. | * to solve the problem, the [[LIgation]] of [[FSC]] and [[Est13]] shall be repeated from the beginning. | ||
* [[Image]] | * [[Image]] | ||
* Additionally,the decision was to go on with the Ligation of [[EstA]] into the Plasmids ([[Est13]] x [[phoA]] x [[pET16b(+)]]) and ([[FSC]] x [[phoA]] x [[pET16b(+)]]), in case that the PCR was proceeded under wrong conditions. | * Additionally,the decision was to go on with the Ligation of [[EstA]] into the Plasmids ([[Est13]] x [[phoA]] x [[pET16b(+)]]) and ([[FSC]] x [[phoA]] x [[pET16b(+)]]), in case that the PCR was proceeded under wrong conditions. | ||
| - | === Friday, 01.06. === | + | ==== Friday, 01.06. ==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of ([[phoA]] x [[pET16b(+)]]) |
** enzymes: NcoI, NdeI | ** enzymes: NcoI, NdeI | ||
** incubation: 1,5 h, 37°C | ** incubation: 1,5 h, 37°C | ||
| Line 735: | Line 735: | ||
** c([[[[phoA]] x [[pET16b(+)]])= 29 ng/µl | ** c([[[[phoA]] x [[pET16b(+)]])= 29 ng/µl | ||
** incubation: over night, 25°C | ** incubation: over night, 25°C | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of ([[FSC]]x ([[PhoA]] x [[pET16b(+)]]) |
* enzymes: NcoI, EcoRI | * enzymes: NcoI, EcoRI | ||
* incubation: 1,5h, 37°C | * incubation: 1,5h, 37°C | ||
| - | == week 8/ | + | === week 8/CW 24 === |
| - | === Monday, 04.06. === | + | ==== Monday, 04.06. ==== |
| - | * restriction of ([[FSC]]x([[PhoA]]x[[pET16b(+)]]) and ([[phoA]]x[[pET16b(+)]]) is cleaned up by [ | + | * restriction of ([[FSC]]x([[PhoA]]x[[pET16b(+)]]) and ([[phoA]]x[[pET16b(+)]]) is cleaned up by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
**[[Image]] | **[[Image]] | ||
**c([[FSC]]x ([[PhoA]] x [[pET16b(+)]]) = 14 ng/µl | **c([[FSC]]x ([[PhoA]] x [[pET16b(+)]]) = 14 ng/µl | ||
*[[Electroporation]] of [[DH5alpha]] with [[Ligation]]: ([[FSC]]x[[PhoA]]x[[pET16b(+)]]), ([[EST13]]x[[PhoA]]x[[pET16b(+)]]) [[Friday, 01.06]] | *[[Electroporation]] of [[DH5alpha]] with [[Ligation]]: ([[FSC]]x[[PhoA]]x[[pET16b(+)]]), ([[EST13]]x[[PhoA]]x[[pET16b(+)]]) [[Friday, 01.06]] | ||
| - | === Tuesday, 05.06. === | + | ==== Tuesday, 05.06. ==== |
*[[colony-PCR]] on [[EST13]] & [[FSC]] is negative. | *[[colony-PCR]] on [[EST13]] & [[FSC]] is negative. | ||
| - | === Wednesday, 06.06. === | + | ==== Wednesday, 06.06. ==== |
* [[Electroporation]] of [[DH5alpha]] with [[Ligation]]-assays from 01.06. | * [[Electroporation]] of [[DH5alpha]] with [[Ligation]]-assays from 01.06. | ||
* [[Ligation]] of [[EstA]][[Wednesday,30.05]] and the Plasmids ([[Est13]]x[[phoA]]x[[pET16b(+)]]),([[FSC]]x[[phoA]]x[[pET16b(+)]]) from [[Monday,04.06.]] | * [[Ligation]] of [[EstA]][[Wednesday,30.05]] and the Plasmids ([[Est13]]x[[phoA]]x[[pET16b(+)]]),([[FSC]]x[[phoA]]x[[pET16b(+)]]) from [[Monday,04.06.]] | ||
| Line 752: | Line 752: | ||
** incubation: 2h, 25°C | ** incubation: 2h, 25°C | ||
**[[Electroporation]] of [[DH5alpha]] with ([[EstA]]x[[Est13]]x[[phoA]]x[[pET16b(+)]]) and ([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]]) | **[[Electroporation]] of [[DH5alpha]] with ([[EstA]]x[[Est13]]x[[phoA]]x[[pET16b(+)]]) and ([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]]) | ||
| - | === Friday, 08.06. === | + | ==== Friday, 08.06. ==== |
* No cells are grown on plates, so the plates are incubated for another two days at RT. | * No cells are grown on plates, so the plates are incubated for another two days at RT. | ||
| - | == week8/ | + | === week8/ CW24 === |
| - | === Monday, 11.06.=== | + | ==== Monday, 11.06.==== |
* all plates are covered with cells, so a [[colony-PCR]] is performed on either [[Est13]],[[FSC]] or [[EstA]] | * all plates are covered with cells, so a [[colony-PCR]] is performed on either [[Est13]],[[FSC]] or [[EstA]] | ||
* primer: | * primer: | ||
| Line 762: | Line 762: | ||
** [[SKV c1 up NcoI]], [[SKV c1 lo EcoRI]] | ** [[SKV c1 up NcoI]], [[SKV c1 lo EcoRI]] | ||
** Every PCR is done in 8 assays à 50 µL, T<sub>A</sub> = 57°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90 s | ** Every PCR is done in 8 assays à 50 µL, T<sub>A</sub> = 57°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90 s | ||
| - | === Tuesday, 12.06. === | + | ==== Tuesday, 12.06. ==== |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] shows, that EstA has been inserted in both Plasmids, ([[Est13]]x[[PhoA]]x[[pET16b(+)]]) and ([[FSC]]x[[PhoA]]x[[pET16b(+)]]) |
| - | * [[ | + | * [[File:TU_Darmstadt_logo.png|200px]] |
* colonies No.3,4 ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]) and No.15,16 ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]) seem to be positive on EstA and are picked to be grown in 5ml DYT/Amp medium. | * colonies No.3,4 ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]) and No.15,16 ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]) seem to be positive on EstA and are picked to be grown in 5ml DYT/Amp medium. | ||
*[[colony-PCR]] on [[Est13]] and [[FSC]] shows a lot of unspecific bands, so cloning of the genes from the beginning has to be dropped. | *[[colony-PCR]] on [[Est13]] and [[FSC]] shows a lot of unspecific bands, so cloning of the genes from the beginning has to be dropped. | ||
| - | **[[ | + | **[[File:TU_Darmstadt_logo.png|200px]] |
| - | === Wednesday, 13.06. === | + | ==== Wednesday, 13.06. ==== |
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the clones No. 3,4,15 and 16. [[Tuesday, 12.06.]] |
** c([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]])= | ** c([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]])= | ||
** No.3=65,17 ng/µl | ** No.3=65,17 ng/µl | ||
| Line 777: | Line 777: | ||
** No.16=59,7 ng/µl | ** No.16=59,7 ng/µl | ||
* for gene-expression, it is performed a [[Elektroporation]] of [[BL21]]-cells with 1µl of the PLasmid-DNA which has just been isolated. | * for gene-expression, it is performed a [[Elektroporation]] of [[BL21]]-cells with 1µl of the PLasmid-DNA which has just been isolated. | ||
| - | === Friday, 15.06.=== | + | ==== Friday, 15.06.==== |
* Transformation of [[BL21]] worked well | * Transformation of [[BL21]] worked well | ||
* [[Tributyrinagar plates]] with 1mM IPTG were generated, to induce the Lac-Promotor of [[pET16b(+)]], and to demonstrate the esterase-activity by the lysis of Tributyrin. | * [[Tributyrinagar plates]] with 1mM IPTG were generated, to induce the Lac-Promotor of [[pET16b(+)]], and to demonstrate the esterase-activity by the lysis of Tributyrin. | ||
| - | * afterwards [[Tributyrinagar plates]] are inoculated with cells which were transformed by [ | + | * afterwards [[Tributyrinagar plates]] are inoculated with cells which were transformed by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] No.3,4,15,16 on [[Wednesday, 13.06.]] |
** incubation: 37°C, 2 days | ** incubation: 37°C, 2 days | ||
| - | == week9/ | + | === week9/CW25 === |
| - | === Monday, 18.06.=== | + | ==== Monday, 18.06.==== |
* [[Tributyrinagar plates]] are covered with cells, but no lysis is visible. | * [[Tributyrinagar plates]] are covered with cells, but no lysis is visible. | ||
** plates are incubated for another day at 25°C. | ** plates are incubated for another day at 25°C. | ||
| - | === Tuesday, 19.06. === | + | ==== Tuesday, 19.06. ==== |
* no lysis, plates stay incubated at 25°C. | * no lysis, plates stay incubated at 25°C. | ||
| - | === Wednesday, 20.06. === | + | ==== Wednesday, 20.06. ==== |
* no lysis | * no lysis | ||
| - | === Friday, 22.06. === | + | ==== Friday, 22.06. ==== |
* The plasmids No.3,4 ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]) and No.15,16 ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]) are prepared for sequencing at [[Seqlab]] | * The plasmids No.3,4 ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]) and No.15,16 ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]) are prepared for sequencing at [[Seqlab]] | ||
* primer: | * primer: | ||
| Line 805: | Line 805: | ||
Sequencing was not successfull. | Sequencing was not successfull. | ||
| - | It was discovered later, that there was a contamination of the [[DH5alpha]] cells with other plasmid-DNA, probably caused by [ | + | It was discovered later, that there was a contamination of the [[DH5alpha]] cells with other plasmid-DNA, probably caused by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation]. |
[[Verlinkung Arne]] | [[Verlinkung Arne]] | ||
Due to this fact, the isolated plasmids were always mixed up with other plasmids, and sequencing had to fail. | Due to this fact, the isolated plasmids were always mixed up with other plasmids, and sequencing had to fail. | ||
| Line 813: | Line 813: | ||
which causes a lysis of the cell-membrane and may have an selective effect on [[FSC]]-negative mutants while cultivating the cells. | which causes a lysis of the cell-membrane and may have an selective effect on [[FSC]]-negative mutants while cultivating the cells. | ||
| - | |||
== BioBricks == | == BioBricks == | ||
=== Week 1 / Kw 29 === | === Week 1 / Kw 29 === | ||
| Line 829: | Line 828: | ||
#: gene of interest: [[pNB-Est13 part2]] for designing part [[BBa_K808026]] via [[SOE PCR]] | #: gene of interest: [[pNB-Est13 part2]] for designing part [[BBa_K808026]] via [[SOE PCR]] | ||
#: primers: [[BBa Est13 down]] & [[SOE Est13 mut up]] | #: primers: [[BBa Est13 down]] & [[SOE Est13 mut up]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] for quality control |
| - | * preparative [ | + | * preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] products |
** 1. PCR (gene of interest: [[phoA]]): c(PhoA)=19 ng/µL | ** 1. PCR (gene of interest: [[phoA]]): c(PhoA)=19 ng/µL | ||
** 2. PCR (gene of interest: [[pNB-Est13 part1]]): c(pNB-Est13 part1)=39 ng/µL | ** 2. PCR (gene of interest: [[pNB-Est13 part1]]): c(pNB-Est13 part1)=39 ng/µL | ||
| Line 845: | Line 844: | ||
==== Wednesday, 18.07.12 ==== | ==== Wednesday, 18.07.12 ==== | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of [[SOE PCR]] (from [[Tuesday, 17.07.12]]) |
[[120718 SOE PCR Est13.jpg]] | [[120718 SOE PCR Est13.jpg]] | ||
| - | * preperative [ | + | * preperative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of SOE PCR produkt |
* [[PCR 1]] | * [[PCR 1]] | ||
: gene of interest: [[EstA]] from [[SKV]] [[Date]] for designing part [[BBa_K808027]] | : gene of interest: [[EstA]] from [[SKV]] [[Date]] for designing part [[BBa_K808027]] | ||
| Line 855: | Line 854: | ||
** T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 75 s | ** T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 75 s | ||
** GC Buffer is used | ** GC Buffer is used | ||
| - | * preparative [ | + | * preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
[[120718 EstA mit bba.jpg]] | [[120718 EstA mit bba.jpg]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] product |
** gene of interest: [[EstA]]: c(pNB-Est13 part2)=119 ng/µL | ** gene of interest: [[EstA]]: c(pNB-Est13 part2)=119 ng/µL | ||
** stored in freezer | ** stored in freezer | ||
==== Thursday, 19.07.12 ==== | ==== Thursday, 19.07.12 ==== | ||
| - | * inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([ | + | * inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC]) |
* [[DNA Digestion]] of the following parts | * [[DNA Digestion]] of the following parts | ||
## [[PhoA]] from [[Tuesday, 17.07.12]] | ## [[PhoA]] from [[Tuesday, 17.07.12]] | ||
| Line 877: | Line 876: | ||
** [[PhoA]] | ** [[PhoA]] | ||
** [[EstA]] | ** [[EstA]] | ||
| - | * [[bacterial transformation]] by [ | + | * [[bacterial transformation]] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [[DH5alpha]] |
==== Friday, 20.07.12 ==== | ==== Friday, 20.07.12 ==== | ||
| - | * the following [ | + | * the following [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] from [[Thursday, 19.07.12]] worked |
**[[Ligation]] into digested and 5' dephosphorylated [[pSB1C3]] | **[[Ligation]] into digested and 5' dephosphorylated [[pSB1C3]] | ||
*** [[pNB-Est13]] | *** [[pNB-Est13]] | ||
*** [[PhoA]] | *** [[PhoA]] | ||
* inoculation of 5 mL [[DYT-media]] with 5 µL CAM and two positive colonies from each transformation (from pNB-Est13 3 colonies are taken) . | * inoculation of 5 mL [[DYT-media]] with 5 µL CAM and two positive colonies from each transformation (from pNB-Est13 3 colonies are taken) . | ||
| - | ** in addition [ | + | ** in addition [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] is already on plate, but to controle it, 2 [[colony PCR]]s are performed |
* [[Ligation]] of [[EstA]] into digested and 5' dephosphorylated [[pSB1C3]] | * [[Ligation]] of [[EstA]] into digested and 5' dephosphorylated [[pSB1C3]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [[DH5alpha]] |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of inoculated FsC in pSB1C3 ([[BBa_K808025]]) from [[Thursday, 19.07.12]] failed |
| - | * inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([ | + | * inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC]) |
** incubation at 37°C over weekend of transformations and picked colonies | ** incubation at 37°C over weekend of transformations and picked colonies | ||
| - | === Week 2 / | + | === Week 2 / CW 30 === |
==== Monday, 23.07.12 ==== | ==== Monday, 23.07.12 ==== | ||
* [[Colony PCR]] of picked colonies from [[Friday, 20.07.12]] | * [[Colony PCR]] of picked colonies from [[Friday, 20.07.12]] | ||
[[120723 BBaPhoA BBa PhoA BBa FsC BBa FsC BBa Est13 BBa Est13 BBa Est13.jpg]] | [[120723 BBaPhoA BBa PhoA BBa FsC BBa FsC BBa Est13 BBa Est13 BBa Est13.jpg]] | ||
| - | * 2-3 [[PhoA]] colonies, 4-5 [ | + | * 2-3 [[PhoA]] colonies, 4-5 [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] colonies, 6-8 [[pNB-Est13]] colonies |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of one inoculated colony in DYT-media with CAM from [[Friday, 20.07.12]] |
** c([[PhoA]] in [[pSB1C3]])=220 ng/µL | ** c([[PhoA]] in [[pSB1C3]])=220 ng/µL | ||
** c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL | ** c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL | ||
| - | * inoculation of 2 x 5 mL [[DYT-media]]-CAM with [[pSB1C3]] carrying [ | + | * inoculation of 2 x 5 mL [[DYT-media]]-CAM with [[pSB1C3]] carrying [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] |
* second transformation of [[EstA]] from [[Friday, 20.07.12]] worked, but too many colonies! | * second transformation of [[EstA]] from [[Friday, 20.07.12]] worked, but too many colonies! | ||
** purity plate is done | ** purity plate is done | ||
==== Tuesday, 24.07.12 ==== | ==== Tuesday, 24.07.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the 2 [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] [[DYT-media]]-CAM cultures with [[pSB1C3]] carrying [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] from yesterday |
| - | ** c([ | + | ** c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=168 ng/µL |
* purity plate of [[EstA]] in [[pSB1c3]] is positive | * purity plate of [[EstA]] in [[pSB1c3]] is positive | ||
** colonies picked | ** colonies picked | ||
| Line 911: | Line 910: | ||
** GC Buffer is used | ** GC Buffer is used | ||
==== Wednesday, 25.07.12 ==== | ==== Wednesday, 25.07.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of [[Colony PCR]] |
** worked out well | ** worked out well | ||
[[120725 Colony BBaEstA positiv probe auf SKV EstA.jpg]] | [[120725 Colony BBaEstA positiv probe auf SKV EstA.jpg]] | ||
| Line 921: | Line 920: | ||
** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VR]], 9 µL ddH<sub>2</sub>O | ** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VR]], 9 µL ddH<sub>2</sub>O | ||
** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VF2]], 9 µL ddH<sub>2</sub>O | ** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VF2]], 9 µL ddH<sub>2</sub>O | ||
| - | ** 7 µL of c([ | + | ** 7 µL of c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=168 ng/µL, 1µL [[VR]], 7 µL ddH<sub>2</sub>O |
| - | ** 7 µL of c([ | + | ** 7 µL of c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=168 ng/µL, 1µL [[VF2]], 7 µL ddH<sub>2</sub>O |
** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VR]], 9 µL ddH<sub>2</sub>O | ** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VR]], 9 µL ddH<sub>2</sub>O | ||
** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VF2]], 9 µL ddH<sub>2</sub>O | ** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VF2]], 9 µL ddH<sub>2</sub>O | ||
| Line 930: | Line 929: | ||
** [[pNB-Est13]] in [[pSB1C3]] | ** [[pNB-Est13]] in [[pSB1C3]] | ||
** [[PhoA]] in [[pSB1C3]] | ** [[PhoA]] in [[pSB1C3]] | ||
| - | ** [ | + | ** [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]] |
** [[EstA]] in [[pSB1C3]] | ** [[EstA]] in [[pSB1C3]] | ||
** Annotation: incubation is done at 37°C over night | ** Annotation: incubation is done at 37°C over night | ||
| Line 936: | Line 935: | ||
* inoculated [[pNB-Est13]] in [[pSB1C3]] from [[Thursday, 26.07.12]] has not grown over night | * inoculated [[pNB-Est13]] in [[pSB1C3]] from [[Thursday, 26.07.12]] has not grown over night | ||
** new colony pcked from plate and incubated in 5 mL [[DYT-media]]-CAM at 37°C over night | ** new colony pcked from plate and incubated in 5 mL [[DYT-media]]-CAM at 37°C over night | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of 3 mL from the following over night [[DYT-media]] cultures from [[Thursday, 26.07.12]] |
** c([[PhoA]] in [[pSB1C3]])=440 ng/µL | ** c([[PhoA]] in [[pSB1C3]])=440 ng/µL | ||
** c([[pNB-Est13]] in [[pSB1C3]])=450 ng/µL | ** c([[pNB-Est13]] in [[pSB1C3]])=450 ng/µL | ||
| - | ** c([ | + | ** c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=90 ng/µL |
| - | === Week 3 / | + | === Week 3 / CW 31 === |
==== Monday, 30.07.12 ==== | ==== Monday, 30.07.12 ==== | ||
| - | * [[bacterial transformation]] by [ | + | * [[bacterial transformation]] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [[DH5alpha]] with c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL from [[Monday, 23.07.12]] |
==== Tuesday, 31.07.12 ==== | ==== Tuesday, 31.07.12 ==== | ||
| - | * no results in sequencing our BioBricks / parts from [[Thursday, 26.07.12]] except for [ | + | * no results in sequencing our BioBricks / parts from [[Thursday, 26.07.12]] except for [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]] |
* new [[Colony PCR]](similar to [[PCR 2]]) is done on | * new [[Colony PCR]](similar to [[PCR 2]]) is done on | ||
** [[PhoA]] in [[pSB1C3]] | ** [[PhoA]] in [[pSB1C3]] | ||
| - | ** [ | + | ** [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]] |
** [[EstA]] in [[pSB1C3]] | ** [[EstA]] in [[pSB1C3]] | ||
** Annotation: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min | ** Annotation: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min | ||
** 1 batch à 50 µL per part | ** 1 batch à 50 µL per part | ||
| - | * no signal on qualitative [ | + | * no signal on qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
** Trouble shooting (see discussion below) | ** Trouble shooting (see discussion below) | ||
| Line 958: | Line 957: | ||
* Symptoms | * Symptoms | ||
| - | ** very much transformants on crossed out plates after [[bacterial transformation]] by [ | + | ** very much transformants on crossed out plates after [[bacterial transformation]] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] |
** at first [[Colony PCR]] is positive | ** at first [[Colony PCR]] is positive | ||
| - | ** [ | + | ** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep]s result in very high yields (exceeding >200 ng/µL) |
** second [[Colony PCR]] is negative | ** second [[Colony PCR]] is negative | ||
** sequencing failed | ** sequencing failed | ||
| - | ** BUT [ | + | ** BUT [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]] |
*** has had not very much transformants | *** has had not very much transformants | ||
*** has had a seuqencing result | *** has had a seuqencing result | ||
| - | *** its [ | + | *** its [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep]s resulted in medium concentrations, settling around 90 ng/µL |
* Diagnosis: | * Diagnosis: | ||
| - | ** our [[bacterial transformation]] is done by [ | + | ** our [[bacterial transformation]] is done by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] |
** therefor we use electroporation cuvettes, stored in DNA precipitating liquids like isopropanol or ethanol after being washed out. | ** therefor we use electroporation cuvettes, stored in DNA precipitating liquids like isopropanol or ethanol after being washed out. | ||
** long before we started these electroporation cuvettes were in use. | ** long before we started these electroporation cuvettes were in use. | ||
** if not washed out carefully, DNA debris (like vectors and Inserts) accumulates in these storage liquids, due to precipitation | ** if not washed out carefully, DNA debris (like vectors and Inserts) accumulates in these storage liquids, due to precipitation | ||
| - | ** when an [ | + | ** when an [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] is performed with one of these cuvettes, probably containing only little DNA debris, the cells get transformed with numbers of different precipitated plasmids |
** these plasmids contain a variety of resistances, reaching from CAM over KAN to Amp, allowing even "wrong" transformants to grow on selective media | ** these plasmids contain a variety of resistances, reaching from CAM over KAN to Amp, allowing even "wrong" transformants to grow on selective media | ||
** after a short period of time, bacterial colonies start to seperate and repel their plasmids, selecting only the most "comfortables" | ** after a short period of time, bacterial colonies start to seperate and repel their plasmids, selecting only the most "comfortables" | ||
| Line 1,000: | Line 999: | ||
#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] of pNB-Est13 ([[BBa_K808026]]) | #: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] of pNB-Est13 ([[BBa_K808026]]) | ||
#: primers: [[BBa Est13 lo]] & [[BBa Est13 mut up]] | #: primers: [[BBa Est13 lo]] & [[BBa Est13 mut up]] | ||
| - | ** Annotation: every [ | + | ** Annotation: every [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] is performed in 3 batches à 50 µL |
** T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 75 s | ** T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 75 s | ||
| - | * due to time pressure there will be no qualitative but only preperative [ | + | * due to time pressure there will be no qualitative but only preperative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | ** [[PhoA]] only on analytical scale but on same [ | + | ** [[PhoA]] only on analytical scale but on same [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] with [[EstA part1]] |
[[120801 soe pcr esta1 pcr phoa.jpg]] | [[120801 soe pcr esta1 pcr phoa.jpg]] | ||
* 1 cut out [[EstA part1]], 2 [[PhoA]] | * 1 cut out [[EstA part1]], 2 [[PhoA]] | ||
| Line 1,010: | Line 1,009: | ||
[[120801 soe pcr esta2.jpg]] | [[120801 soe pcr esta2.jpg]] | ||
* cut out [[EstA part2]] | * cut out [[EstA part2]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of [[EstA part1]], [[EstA part2]], [[pNB-Est13 part1]] and [[pNB-Est13 part2]] |
** c([[EstA part1]])=19 ng/µL | ** c([[EstA part1]])=19 ng/µL | ||
** c([[EstA part2]])=110 ng/µL | ** c([[EstA part2]])=110 ng/µL | ||
| Line 1,027: | Line 1,026: | ||
** T<sub>A2</sub> = 57°C, t<sub>A2</sub> = 30 s, t<sub>E2</sub> = 75 s | ** T<sub>A2</sub> = 57°C, t<sub>A2</sub> = 30 s, t<sub>E2</sub> = 75 s | ||
** stored over night at 10°C | ** stored over night at 10°C | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
[[120801 SOE PCR Est13 EstA EstApart2 kontrolle.jpg]] | [[120801 SOE PCR Est13 EstA EstApart2 kontrolle.jpg]] | ||
* 2 [[pNB-Est13]], 3 [[ESTA]], 4[[EstA part2]] | * 2 [[pNB-Est13]], 3 [[ESTA]], 4[[EstA part2]] | ||
| Line 1,054: | Line 1,053: | ||
** colonies picked to inoculate 5 ml [[DYT-media]]-CM | ** colonies picked to inoculate 5 ml [[DYT-media]]-CM | ||
==== Saturday, 04.08.12 ==== | ==== Saturday, 04.08.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of picked colonies |
** c([[PhoA]] in [[pSB1C3]])=227 ng/µL | ** c([[PhoA]] in [[pSB1C3]])=227 ng/µL | ||
** c([[pNB-Est13]] in [[pSB1C3]])=148ng/µL | ** c([[pNB-Est13]] in [[pSB1C3]])=148ng/µL | ||
| Line 1,062: | Line 1,061: | ||
: T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s | : T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s | ||
** XylH in [[pSB1C3]] serves as a control in digestion and [[PCR 2]] | ** XylH in [[pSB1C3]] serves as a control in digestion and [[PCR 2]] | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] 1% agarose |
[[120804 Verdau PhoA, EstA, Est13, PCR auf EstA, Est 13, PhoA, Kontrolle auf Insert und Vektor.jpg]] | [[120804 Verdau PhoA, EstA, Est13, PCR auf EstA, Est 13, PhoA, Kontrolle auf Insert und Vektor.jpg]] | ||
* digestion: 2 [[PhoA]], 3 [[EstA]], 4 [[pNB-Est13]] PCR: 6 [[EstA]] with BBa primers, 7 [[EstA]] with [[VR]] & [[VF2]], 8 [[pNB-Est13]] with BBa primers, 10 [[pNB-Est13]] with [[VR]] & [[VF2]], 11 [[PhoA]] with BBa primers, 12 [[PhoA]] with [[VR]] & [[VF2]] | * digestion: 2 [[PhoA]], 3 [[EstA]], 4 [[pNB-Est13]] PCR: 6 [[EstA]] with BBa primers, 7 [[EstA]] with [[VR]] & [[VF2]], 8 [[pNB-Est13]] with BBa primers, 10 [[pNB-Est13]] with [[VR]] & [[VF2]], 11 [[PhoA]] with BBa primers, 12 [[PhoA]] with [[VR]] & [[VF2]] | ||
| Line 1,088: | Line 1,087: | ||
** [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] digested with EcoRI-HF and BsaI-HF in a 50 µL batch | ** [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] digested with EcoRI-HF and BsaI-HF in a 50 µL batch | ||
** [[BsaI-Myctag-EstA- bba suffix]] digested with BsaI-HF and PstI-HF in a 50 µL batch | ** [[BsaI-Myctag-EstA- bba suffix]] digested with BsaI-HF and PstI-HF in a 50 µL batch | ||
| - | * preparative [ | + | * preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
* Failure due to overlooked additional PstI site in GENEART Vectors | * Failure due to overlooked additional PstI site in GENEART Vectors | ||
* inoculation of 5 ml [[DYT-media]]-CAM with retransformations from [[Saturday, 04.08.12]] | * inoculation of 5 ml [[DYT-media]]-CAM with retransformations from [[Saturday, 04.08.12]] | ||
| Line 1,106: | Line 1,105: | ||
* inoculated 5 mL cultures from [[Monday, 06.08.12]] are stored at 4°C | * inoculated 5 mL cultures from [[Monday, 06.08.12]] are stored at 4°C | ||
==== Wednesday, 08.08.12 ==== | ==== Wednesday, 08.08.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of following 5 mL cultures |
** from [[Monday, 06.08.12]] | ** from [[Monday, 06.08.12]] | ||
*** c([[PhoA]] in [[pSB1C3]])=90 ng/µL | *** c([[PhoA]] in [[pSB1C3]])=90 ng/µL | ||
| Line 1,129: | Line 1,128: | ||
*** BsaI and SpeI are used | *** BsaI and SpeI are used | ||
*** digestion for 3 h | *** digestion for 3 h | ||
| - | * preperative [ | + | * preperative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
[[120808 Restriktion mit E BsaI PhoA Est13 und bsai s EstA.jpg]] | [[120808 Restriktion mit E BsaI PhoA Est13 und bsai s EstA.jpg]] | ||
* 3-4 cut out [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]], 5-6 cut out [[BsaI-Myctag-EstA- bba suffix]] | * 3-4 cut out [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]], 5-6 cut out [[BsaI-Myctag-EstA- bba suffix]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
** c([[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]])= 8 ng/µL | ** c([[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]])= 8 ng/µL | ||
** c([[BsaI-Myctag-EstA- bba suffix]])= 26 ng/µL | ** c([[BsaI-Myctag-EstA- bba suffix]])= 26 ng/µL | ||
| Line 1,151: | Line 1,150: | ||
** one colony per plate (1:5:5 & 1:10:10 at room temperature and 1:5:5 & 1:10:10 at room 4°C)is picked for inoculation of 5 mL [[DYT-media]]-CAM | ** one colony per plate (1:5:5 & 1:10:10 at room temperature and 1:5:5 & 1:10:10 at room 4°C)is picked for inoculation of 5 mL [[DYT-media]]-CAM | ||
==== Saturday, 11.08.12 ==== | ==== Saturday, 11.08.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of picked colonies from yesterday |
** testing ligation by [[DNA Digestion]] of plasmids with [[EcorI-HF]] & [[PstI-HF]] | ** testing ligation by [[DNA Digestion]] of plasmids with [[EcorI-HF]] & [[PstI-HF]] | ||
* [[Colony PCR]] on picked colonies | * [[Colony PCR]] on picked colonies | ||
| Line 1,159: | Line 1,158: | ||
[[120811 PCR (VR,VF2) und Verdau (EcoRI,PstI) auf BBa Gesamt von Geneart.jpg]] | [[120811 PCR (VR,VF2) und Verdau (EcoRI,PstI) auf BBa Gesamt von Geneart.jpg]] | ||
* 2-5 [[bba prefix-PhoA-His6tag-pNBEst13-Myctag-EstA- bba suffix]] running at round about 3 kb | * 2-5 [[bba prefix-PhoA-His6tag-pNBEst13-Myctag-EstA- bba suffix]] running at round about 3 kb | ||
| - | === Week 5 / | + | === Week 5 / CW 33 === |
==== Monday, 13.08.12 ==== | ==== Monday, 13.08.12 ==== | ||
* Preparation for designing part [[BBa_K808032]] which is an arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA | * Preparation for designing part [[BBa_K808032]] which is an arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA | ||
| - | * in order to add RBS upstream of PhoA of [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] the following [ | + | * in order to add RBS upstream of PhoA of [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] the following [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR]s are performed |
# [[PCR 1]] on [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]], as a template the pure synthesis product is used | # [[PCR 1]] on [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]], as a template the pure synthesis product is used | ||
#: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min | #: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min | ||
| Line 1,172: | Line 1,171: | ||
#: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min | #: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min | ||
#: primers: [[XbaI Rbs Phoa up]] & [[BBa EstA down]] | #: primers: [[XbaI Rbs Phoa up]] & [[BBa EstA down]] | ||
| - | ** Annotation: each [ | + | ** Annotation: each [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] is performed in 4 batches à 50 µL |
| - | * 2 qualitative [ | + | * 2 qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
[[120813 PCR 1-4 RBSPE 5-8 EXN-Myc-Est 9-12.jpg]] | [[120813 PCR 1-4 RBSPE 5-8 EXN-Myc-Est 9-12.jpg]] | ||
* 2-5 [[bba prefix-RBS-PhoA-His6tag-pNBEst13-BsaI]], 6-9 [[BsaI-Myctag-EstA-bba suffix]] | * 2-5 [[bba prefix-RBS-PhoA-His6tag-pNBEst13-BsaI]], 6-9 [[BsaI-Myctag-EstA-bba suffix]] | ||
[[120813 PCR Q5 auf bba gesamt konstrukt.jpg]] | [[120813 PCR Q5 auf bba gesamt konstrukt.jpg]] | ||
* 2-4 [[bba prefix-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA-bba suffix]] | * 2-4 [[bba prefix-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA-bba suffix]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR]s succeeded |
* [[PCR clean up]] of 1.-3. PCR | * [[PCR clean up]] of 1.-3. PCR | ||
* [[DNA Digestion]] of [[pSB1C3]] carrying [[RFP]] part [[????]] | * [[DNA Digestion]] of [[pSB1C3]] carrying [[RFP]] part [[????]] | ||
| Line 1,186: | Line 1,185: | ||
** enzymes used: [[SpeI]] & [[PstI]] | ** enzymes used: [[SpeI]] & [[PstI]] | ||
** performed in 100 µL batch | ** performed in 100 µL batch | ||
| - | * preparative [ | + | * preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
** c(1.PCR)= 147 ng/µL | ** c(1.PCR)= 147 ng/µL | ||
** c(2.PCR)= 133 ng/µL | ** c(2.PCR)= 133 ng/µL | ||
| Line 1,219: | Line 1,218: | ||
** primers: [[VR]] & [[VF2]] | ** primers: [[VR]] & [[VF2]] | ||
==== Thursday 16.08.12 ==== | ==== Thursday 16.08.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of colonies from yesterday |
* [[DNA Digestion]] of preped colonies with [[EcorI-HF]] & [[PstI-HF]] | * [[DNA Digestion]] of preped colonies with [[EcorI-HF]] & [[PstI-HF]] | ||
** incubation for 1.5 h at 37°C | ** incubation for 1.5 h at 37°C | ||
| - | * qualitative [ | + | * qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
** FAILURE, because bands on gel are inconsistent | ** FAILURE, because bands on gel are inconsistent | ||
[[120816 Testrestriktion der pSB1C3 mit e und p colony platten 1 - 6.jpg]] | [[120816 Testrestriktion der pSB1C3 mit e und p colony platten 1 - 6.jpg]] | ||
| Line 1,252: | Line 1,251: | ||
* when digested with same enzymes of ligations into part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 2, bands are shown at round about 1 - 1.5 kb | * when digested with same enzymes of ligations into part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 2, bands are shown at round about 1 - 1.5 kb | ||
* this leads to our concluison that [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1 was the not digested DNA band, because resulting gene length of cut insert correlates with length of [[BBa_K808000]] ([[Arabinose inducible promotor]]) | * this leads to our concluison that [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1 was the not digested DNA band, because resulting gene length of cut insert correlates with length of [[BBa_K808000]] ([[Arabinose inducible promotor]]) | ||
| - | * for a sharper solution an [ | + | * for a sharper solution an [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] with 0.5 % agarose is performed for digestions of colonies 1.1, 3.1, 4.1, 4.2 plus batches from [[Colony PCR]] from [[Wednesday, 15.08.12]] |
[[120816 Colony 1.1 3.1 4.1 4.2 Colony Pcr 1-8.tif]] | [[120816 Colony 1.1 3.1 4.1 4.2 Colony Pcr 1-8.tif]] | ||
* 2-5 colonies 1.1, 3.1, 4.1, 4.2, 6 - 13 colony 1.1, 2.1, 2.2, 3.1, 4.1, 4.2, 5.1, 5.2 | * 2-5 colonies 1.1, 3.1, 4.1, 4.2, 6 - 13 colony 1.1, 2.1, 2.2, 3.1, 4.1, 4.2, 5.1, 5.2 | ||
| Line 1,261: | Line 1,260: | ||
** colony 5.1, 5.2, 2.1, 2.2 are only carrying part [[[[BBa_K808000]] which encodes for the arabinosis inducable promotor | ** colony 5.1, 5.2, 2.1, 2.2 are only carrying part [[[[BBa_K808000]] which encodes for the arabinosis inducable promotor | ||
** but colony 1.1, 2.1, 2.2 are carrying 1.PCR & 2.PCR or 3.PCR in [[pSB1C3]] | ** but colony 1.1, 2.1, 2.2 are carrying 1.PCR & 2.PCR or 3.PCR in [[pSB1C3]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of named colonies in liquid DYT-culture from [[Thursday 16.08.12]] |
** half culture is preped, half culture serves for [[10% DMSO stocks]] | ** half culture is preped, half culture serves for [[10% DMSO stocks]] | ||
** c(1.1: [[BBa_K808030]])= 81 ng/µL | ** c(1.1: [[BBa_K808030]])= 81 ng/µL | ||
| Line 1,273: | Line 1,272: | ||
** [[BBa_K808000]] (colony 1.1, 4.1, 4.2), with EcoRI / SpeI, serves as upstream part | ** [[BBa_K808000]] (colony 1.1, 4.1, 4.2), with EcoRI / SpeI, serves as upstream part | ||
** [[BBa_K808030]] (colony 5.1, 5.2, 2.1, 2.2), with EcoRI / XbaI, serves as downstream part | ** [[BBa_K808030]] (colony 5.1, 5.2, 2.1, 2.2), with EcoRI / XbaI, serves as downstream part | ||
| - | * preperative [ | + | * preperative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] |
** downstream part | ** downstream part | ||
** c(1.1: [[BBa_K808030]])= 21.5 ng/µL | ** c(1.1: [[BBa_K808030]])= 21.5 ng/µL | ||
| Line 1,297: | Line 1,296: | ||
** colony 5.1 | ** colony 5.1 | ||
| - | === Week 6 / | + | === Week 6 / CW 34 === |
==== Monday 20.08.12 ==== | ==== Monday 20.08.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of incoucaltes colonies from [[Friday, 17.08.12]] |
** c(1.1: [[BBa_K808030]])= 54 ng/µL | ** c(1.1: [[BBa_K808030]])= 54 ng/µL | ||
** c(4.1: [[BBa_K808030]])= 74 ng/µL | ** c(4.1: [[BBa_K808030]])= 74 ng/µL | ||
| Line 1,321: | Line 1,320: | ||
: T<sub>A</sub> = 55°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 5 min | : T<sub>A</sub> = 55°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 5 min | ||
: primers: [[VF2]] & [[VR]] | : primers: [[VF2]] & [[VR]] | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] |
** did not work due to immense gene legth ( around 4 kb) | ** did not work due to immense gene legth ( around 4 kb) | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] for testing is planned |
** inoculation of 5 mL [[DYT-media]]-CAM with colony E,F,C,D | ** inoculation of 5 mL [[DYT-media]]-CAM with colony E,F,C,D | ||
==== Wednesday, 22.08.12 ==== | ==== Wednesday, 22.08.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of inoculated 5 mL [[DYT-media]]-CAM with colony E,F,C,D |
** c(colony C 1.1/5.1 ligated at 4°C)=46 ng/µL | ** c(colony C 1.1/5.1 ligated at 4°C)=46 ng/µL | ||
** c(colony D 1.1/5.1 ligated at 37°c)=46 ng/µL | ** c(colony D 1.1/5.1 ligated at 37°c)=46 ng/µL | ||
| Line 1,333: | Line 1,332: | ||
* [[DNA Digestion]] of 15 µL of preped plasmids with [[EcoRI-HF]] & [[PstI-HF]] | * [[DNA Digestion]] of 15 µL of preped plasmids with [[EcoRI-HF]] & [[PstI-HF]] | ||
** expected insert length: 4.4 kb ([[BBa_K808000]]+[[BBa_K808030]]) | ** expected insert length: 4.4 kb ([[BBa_K808000]]+[[BBa_K808030]]) | ||
| - | *[ | + | *[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] with 0.8% agarose |
[[Verdau vom Gesamtkonstrukt 4.2_2.2 und 5.1_1.1 beide temperaturen.tif]] | [[Verdau vom Gesamtkonstrukt 4.2_2.2 und 5.1_1.1 beide temperaturen.tif]] | ||
* all bands are in same heights, so ligation worked well | * all bands are in same heights, so ligation worked well | ||
* ligation worked well, bands are in estimated length | * ligation worked well, bands are in estimated length | ||
| - | * due to rather bad results of [ | + | * due to rather bad results of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] colonies are used for inoculation of 5 mL [[DYT-media]]-CAM again |
** incubation over night at 37°C | ** incubation over night at 37°C | ||
* evaluation of sequencing from [[Wednesday, 08.08.12]] | * evaluation of sequencing from [[Wednesday, 08.08.12]] | ||
| Line 1,351: | Line 1,350: | ||
** primers [[BBa Est13 up]] & [[BBa Est13 down]] | ** primers [[BBa Est13 up]] & [[BBa Est13 down]] | ||
** performed in 3 batches à 50 µL | ** performed in 3 batches à 50 µL | ||
| - | * analytical [ | + | * analytical [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] 2% agarose |
* [PCR Clean up]] | * [PCR Clean up]] | ||
* [[DNA Digestion]] of PCR product with [[EcoRI-HF]] & [[PstI-HF]] over night at 37°C | * [[DNA Digestion]] of PCR product with [[EcoRI-HF]] & [[PstI-HF]] over night at 37°C | ||
==== Thursday, 23.09.12 ==== | ==== Thursday, 23.09.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of colonies from |
** c(colony C 1.1/5.1 ligated at 4°C)=147 ng/µL | ** c(colony C 1.1/5.1 ligated at 4°C)=147 ng/µL | ||
** c(colony D 1.1/5.1 ligated at 37°c)=131ng/µL | ** c(colony D 1.1/5.1 ligated at 37°c)=131ng/µL | ||
** c(colony E 4.2/2.2 ligated at 4°C)=85 ng/µL | ** c(colony E 4.2/2.2 ligated at 4°C)=85 ng/µL | ||
** c(colony F 4.2/2.2 ligated at 37°C)=128 ng/µL | ** c(colony F 4.2/2.2 ligated at 37°C)=128 ng/µL | ||
| - | * preparative [ | + | * preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of digested [[pNB-Est13]] from yesterday |
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] leads to c([[pNB-Est13]]- cut E/P)=25 ng/µL |
* [[Ligation]] of digested [[pNB-Est13]] in digested [[pSB1C3]] | * [[Ligation]] of digested [[pNB-Est13]] in digested [[pSB1C3]] | ||
** 1:5 ratio is used | ** 1:5 ratio is used | ||
| Line 1,370: | Line 1,369: | ||
* sequencing of | * sequencing of | ||
** [[BBa_K808032]] (colony C 1.1/5.1 ligated at 4°C), primers [[VF2]], [[XbaI Rbs Phoa up]], [[BBa Ara Promo lo]], [[SE Est13 mut up]], [[SOE Est13 mut lo]], [[BBa EstA down]], [[VR]], 1.5 µL each, | ** [[BBa_K808032]] (colony C 1.1/5.1 ligated at 4°C), primers [[VF2]], [[XbaI Rbs Phoa up]], [[BBa Ara Promo lo]], [[SE Est13 mut up]], [[SOE Est13 mut lo]], [[BBa EstA down]], [[VR]], 1.5 µL each, | ||
| - | === Week 7 / | + | === Week 7 / CW 35 === |
==== Monday, 27.08.12 ==== | ==== Monday, 27.08.12 ==== | ||
| - | * [ | + | * [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of picked colony from [[BBa_K808026]] ([[pNB-Est13]] in [[pSB1C3]]) |
** c([[pNB-Est13]] in [[pSB1C3]])=116 ng/µL | ** c([[pNB-Est13]] in [[pSB1C3]])=116 ng/µL | ||
* sequencing of [[pNB-Est13]] in [[pSB1C3]] | * sequencing of [[pNB-Est13]] in [[pSB1C3]] | ||
| Line 1,381: | Line 1,380: | ||
*** stop incubation at at OD<sub>600</sub>=0.7 | *** stop incubation at at OD<sub>600</sub>=0.7 | ||
*** storaging cultures on ice for 15mins | *** storaging cultures on ice for 15mins | ||
| - | *** inducing with ~ 1.5% L-arabinose (using 3.75 mL of 20% mw | + | *** inducing with ~ 1.5% L-arabinose (using 3.75 mL of 20% mw L-arobinose solution) |
*** incubation at 20°C, 25°C and 30°C over night | *** incubation at 20°C, 25°C and 30°C over night | ||
==== Tuesday, 28.08.12 ==== | ==== Tuesday, 28.08.12 ==== | ||
| Line 1,405: | Line 1,404: | ||
* running 2 [[SDS-Laemmli gel]]s | * running 2 [[SDS-Laemmli gel]]s | ||
** pellet is treated with n-Dodecyl-ß-maltoside (DDM) in order to solve membrane proteins | ** pellet is treated with n-Dodecyl-ß-maltoside (DDM) in order to solve membrane proteins | ||
| - | [[ | + | [[File:TU_Darmstadt_logo.png|200px]] |
* one gel is used to perform a second [[Western blot]] with a myc positive probe | * one gel is used to perform a second [[Western blot]] with a myc positive probe | ||
[[120831 Westermblot 2 auf Gesamt konstrukt aus DH5 alpha 5111.tif]] | [[120831 Westermblot 2 auf Gesamt konstrukt aus DH5 alpha 5111.tif]] | ||
Revision as of 02:14, 19 September 2012
WORK IN PROGRESS DO NO INTERFERE!!!
Contents
|
Degradation
This page features the work carried out by the degradation team. The main objectives were the production of a BioBrick containing Fusarium solani cutinase or Est13 esterase two enzymes potentially enabling E.coli of PET degradation and over-expression stems for activity screening.
Activity tests of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
Week 1 / CW 35
Friday, 31.08.12
- Activity assay in DYT with DH5α containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] (arabinose inducable RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
| Component | test tube 1 | test tube 2 | test tube 3 | test tube 4 |
|---|---|---|---|---|
| DYT-medium | 8 mL | 8 mL | 8 mL | 8 mL |
| PET particle | yes | yes | yes | yes |
| bacteria | yes | yes | yes | no |
| induced | 1.5% L-arabinose | 1.5% L-arabinose | no | no |
- test seemed to have worked: but an induced test tube without PET-granula was missing
Week 2 / CW 36
Tuesday, 04.09.12
- Activity assay in DYT with DH5α containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
| Component | test tube 1 | test tube 2 | test tube 3 | test tube 4 | test tube 5 |
|---|---|---|---|---|---|
| DYT-medium | 8 mL | 8 mL | 8 mL | 8 mL | 8 mL |
| PET particle | yes | yes | no | yes | yes |
| bacteria | yes | yes | yes | no | yes |
| induced | 1.5% L-arabinose | 1.5% L-arabinose | 1.5% L-arabinose | no | no |
- looks good but test tube 2 shows no significant change of colour
Wednesday, 05.09.12
- Activity assay in DYT with DH5α containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
| Component | tube 1 | tube 2 | tube 3 | tube 4 | tube 5 | tube 6 | tube 7 | tube 8 | tube 9 |
|---|---|---|---|---|---|---|---|---|---|
| DYT-medium | 8 mL | 8 mL | 8 mL | 8 mL | 8 mL | 8 mL | 8 mL | 8 mL | 8 mL |
| PET particle | yes | yes | yes | yes | no | no | yes | yes | yes |
| bacteria | yes | yes | yes | yes | yes | yes | yes | yes | no |
| induced | 1.5% L-arabinose | 1.5% L-arabinose | 1.5% L-arabinose | 1.5% L-arabinose | 1.5% L-arabinose | 1.5% L-arabinose | no | no | no |
- all induced tubes turned yellow, even without PET-granula as a substrate
- no more avtivity tests, we are awaiting the evaluation of test expression series of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 arabinose inducible promoter] conjugated to GFP
Trouble shooting
- evaluation shows a very high sensisty of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 arabinose inducible promoter [BBa_K808000]] even to low concentrations of L-arabinose (expression starts at around 0.01%)
- induction of the DH5α with 1.5% arabinose ended fatal due to extrem expression of the transmembrane construct [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 RBS-PhoA-His6tag-pNBEst13-Myctag-EstA]
- starting test expressions with lower L-arabinose concentrations ranging from 0.05% - 1%
Team II Kill Curves
Thursday, 06.09.12
- Activity assay in DYT with DH5α containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 [BBa_K808032]] (arabinose inducable RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
| Component | tube 1 | tube 2 | tube 3 | tube 4 | tube 5 | tube 6 | tube 7 | tube 8 | tube 9 | tube 10 | tube 11 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| DYT-medium | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL |
| PET particle | yes | yes | yes | yes | yes | yes | no | no | no | no | no |
| bacteria | yes | yes | yes | yes | yes | yes | yes | yes | yes | yes | no |
| induced | 1% L-arabinose | 1% L-arabinose | 0.75% L-arabinose | 0.75% L-arabinose | no | no | 1% L-arabinose | 1% L-arabinose | 0.75% L-arabinose | 0.75% L-arabinose | no |
- all induced tubes turned yellow, even without PET-granula as a substrate
Week 3 / CW 37
Monday, 10.09.12
- Activity assay in DYT with DH5α containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 [BBa_K808032]] (arabinose inducable RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
| Component | tube 1 | tube 2 | tube 3 | tube 4 | tube 5 | tube 6 | tube 7 | tube 8 | tube 9 | tube 10 | tube 11 | tube 12 | tube 13 | tube 14 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| DYT-medium | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL | 5 mL |
| PET particle | no | no | no | no | yes | yes | yes | yes | no | no | no | no | yes | yes |
| PET stripe | yes | yes | yes | yes | no | no | no | no | no | no | no | no | no | no |
| bacteria | yes | yes | yes | yes | yes | yes | yes | yes | yes | yes | yes | yes | yes | no |
| induced | 0.05% L-arabinose | 0.1% L-arabinose | 0.2% L-arabinose | 0.4% L-arabinose | 0.05% L-arabinose | 0.1% L-arabinose | 0.2% L-arabinose | 0.4% L-arabinose | 0.05% L-arabinose | 0.1% L-arabinose | 0.2% L-arabinose | 0.4% L-arabinose | no | no |
- test tube 14: DYT without bacteria contains CAM, Kan, AMP
- 0.05% induced tubes with substrate show difference to tube without substrate
- quantification is possible via meassurement of absorption
- activity assay on LB-Tributyrin-CAM-plates with L-arabinose concentrations: 0.1%, 0.2%, 0.4%
Protein Expression
CW 24
Thursday, 14.06.2012
- PCR for protein expression of FsC
- each PCR is performed in 5 batches à 50 µL.
- Parameter: TA = 57°C, tA = 35 s, tE = 65 s
- PCR on pEST100 vector for expression with pEX vector
- gene of interest: FsC for designed part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
- primers: pEX FsC His SfiI up and pEX FsC Stop SfiI lo
Friday, 15.06.2012
- Agarose gel electrophoresis for quality control
CW 25
Wednesday, 20.06.2012
- Agarose gel electrophoresis for preparation
- Gel extraction of PCR products
- Concentration of produced FsC sequence: 40 ng/µL
Thursday, 21.06.2012
- Restriction digest of PCR product from 14.06.
- Enzymes used: SfiI
- NEBuffer: 4
- Digestion time: over night
- Digestion temperature: 50°C
Friday, 22.06.2012
- Miniprep of pEX vector
- Concentration range: 240-488 ng/µL
- Restriction digest of pEX
- Enzymes used: SfiI
- NEBuffer: 4
- Digestion time: 3 days
- Digestion temperature: 50°C
CW 26
Monday, 25.06.2012 =
- Agarose gel electrophoresis for quality control
- Gel extraction of pEX and digested FsC sequence from 21.06.
- Ligation of pEX with FsC sequence with the ratio 1:3 and 1:5
| Component | 1:3 | 1:5 |
|---|---|---|
| FsC sequence | 1.12 µL | 2.2 µL |
| pEX | 0.64 µL | 0.64 µL |
| Ligase buffer | 4 µL | 4 µL |
| T4 DNA ligase | 1 µL | 1 µL |
| H2O | 33 µL | 30 µL |
- Ligation time: over night
Tuesday, 26.06.2012
- Bacterial transformation by Electroporation of TOP10 with the ligation product pEX-FsC from 25.06.
Wednesday, 27.06.2012
Thursday, 28.06.2012
- Miniprep of pEX-Fsc in TOP10
- Concentration: 45 ng/µL and 405 ng/µL
- Bacterial transformation by Electroporation of BmH7118 with the Miniprep product pEX-FsC
CW 27
Monday, 02.07.2012
- Protein expression of FsC at different temperatures (16°C, 25°C, 30°C and 37°C) for the final step of the expression
Tuesday, 03.07.2012
- Purification of Periplasmatic Proteins for all expression temperatures
Wednesday, 04.07.2012
- Protein purification without cell disrupter
- SDS-PAGE (Schägger) analysis of all collected samples
| Expression at 16°C | Expression at 25°C |
|---|---|
| |
|
| Expression at 30°C | Expression at 37°C |
| |
|
- The best results were maintained at an expression temperature of 30°C
Friday, 06.07.2012
CW 28
Monday, 09.07.2012
- Miniprep of pEX-FsC from 06.07.
- Concentration: 470 ng/µL
- Preparation for sequencing at Eurofins
- Barcode 043 pEX-FsC with primer M13 Reverse up
- Barcode 044 pEX-FsC with primer ClaI pIII lo
- Plasmid DNA: 5µL
- Primer: 3µL
- H2O: 7µL
Wednesday, 11.07.2012
- Protein expression of FsC according to standard protocol
Thursday, 12.07.2012
- Protein purification by standard protocol
- SDS-PAGE (Schägger) analysis of samples
CW 29
Monday, 16.07.2012
- Protein expression of FsC according to standard protocol
Tuesday, 17.07.2012
- Protein purification by standard protocol
- SDS-PAGE (Schägger) analysis of samples
CW 31
Monday, 30.07.2012
- Protein expression of FsC according to standard protocol
Tuesday, 31.07.2012
- Protein purification by standard protocol
- SDS-PAGE (Schägger) analysis of samples
SOE PCR
Week 1 / CW 17
Tuesday, 24.04.12
- Production of electrocompetent cells DH5alpha and BL21
- Pouring of LB-Agar plates with ampecilin resistance (AMP)
- setting of DYT media
- Electroporation of BL21 with the following plasmids
Wednesday, 25.04.12
- Miniprep of the 3 overnigth Bl21 cultures and concentration meassurement via Nanodrop
Thursday, 26.04.12
Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE = 25 s
- PCR on pEST100
- gene of interest: Promo-LacO-RBS-Phoa for SKV
- primers: SKV a1 up XbaI & SKV a1 lo NdeI
- PCR on pEST100
- gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
- primers: SOE A up & SOE a1 lo
- PCR on pEST100
- gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with FsC
- primers: SOE A up & SOE a2 lo
- PCR on pEST100
- gene of interest: FsC for SOE PCR with Promo-LacO-RBS-Phoa and EstA part1
- primers: SOE b2 up & SOE b2 lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
- primers: SOE b1 up & SOE Est13 mut lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
- primers: SOE Est13 mut up & SOE b1 lo
- Agarose gel electrophoresis (Agorese gele elektrophoresis) for qualitiy control
Friday, 27.04.12
- clean up of 1. PCR on pEST100 from Thursday, 26.04.12 via Ammonium acetate - Ehtanol DNA precipitation, solved in 54 µl ddH2O
- restriction of cleaned up 1. PCR and pET16b with XbaI and NdeI over weekend at 37°C
Week 2 / CW 18
Monday, 30.04.12
- SOE PCR
- pNB-Est13 part1 & pNB-Est13 part2, primers: SOE b1 up & SOE b1 lo
- Promo-LacO-RBS-Phoa & FsC, primers: SOE A up & SOE b2 lo
- Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE1 = 20s, tE1 = 35s
- Agarose gel electrophoresis of SOE PCR
- SOE PCR of Promo-LacO-RBS-Phoa-FsC worked, pNB-Est13 did not due to missing clean up via Agarose gel electrophoresis, precipitation is insufficient, we do it again
- PCRs
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
- primers: SOE b1 up & SOE Est13 mut lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
- primers: SOE Est13 mut up & SOE b1 lo
- Agarose gel electrophoresis
- 1. PCR worked well 2. PCR did not
Wednesday, 02.05.12
- PCRs
- PCR on pEST100
- gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
- primers: SOE A up & SOE a1 lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
- primers: SOE b1 up & SOE Est13 mut lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
- primers: SOE Est13 mut up & SOE b1 lo
- TA = 57°C, ta = 35s, tE = 25 s
- every PCR is performed in 3 batches à 50 µL
- Promega gel extraction
- c(1.PCR)=6 ng/µL
- c(2.PCR)=12 ng/µL
- c(3.PCR)=13 ng/µL
- SOE PCR of 2.PCR and 3.PCR in order to form pNB-Est13 for SOE PCR with Promo-LacO-RBS-Phoa and EstA
- TA1 = 60°C, ta1 = 25 s, tE1 = 25 s
- TA = 60°C, ta2 = 25 s, tE2 = 35 s
Tuesday, 03.05.12
- preparative Agarose gel electrophoresis of SOE PCR from yesterday
- Gel extraction
- cpNB-Est13 7 ng/µL
Friday, 04.05.12
- PCRs
- PCR on pEST100
- gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
- primers: SOE A up & SOE a1 lo
- PCR on pEST100
- gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13
- primers: SOE c1 up & SOE EstA mut PstI out lo
- PCR on pEST100
- gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and FsC
- primers: SOE c2 up & SOE EstA mut PstI out lo
- PCR on pEST100
- gene of interest: EstA part2 for SOE PCR with EstA part1
- primers: SOE EstA mut PstI out up & SOE D lo
- annotation: TA = 60°C, ta = 25 s, tE = 35 s
- every PCR is performed in 3 batches à 50 µL
- preparative Agarose gel electrophoresis
Week 3 / CW 19
Tuesday 08.05.12
- gene of interest: Promo-LacO-RBS-Phoa-pNBEst13 for SOE PCR with EstA
- primers: SOE A up & SOE b1 lo
- template: pNB-Est13 from last friday & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12
- annotation: TA1 = 60°C, ta1 = 25 s, tE1 = 35 s
- TA2 = 60°C, ta2 = 25 s, tE2 = 1 min
- did not work
Wednesday, 09.05.12
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
- primers: SOE b1 up & SOE Est13 mut lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
- primers: SOE Est13 mut up & SOE b1 lo
- PCR on pEST100
- gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13
- primers: SOE c1 up & SOE EstA mut PstI out lo
- PCR on pEST100
- gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and FsC
- primers: SOE c2 up & SOE EstA mut PstI out lo
- PCR on pEST100
- gene of interest: EstA part2 for SOE PCR with EstA part1
- primers: SOE EstA mut PstI out up & SOE D lo
- annotation: TA = 60°C, ta = 25 s, tE = 35 s
- every PCR is performed in 2 batches à 50 µL
Friday, 09.05.12
- qualitative Agarose gel electrophoresis
-
- both EstA part 1 (3.PCR & 4.PCR) did not work well
Week 4 / CW 20
Monday, 14.05.12
- Gel extraction of remaining PCRs from Wednesday, 09.05.12
- c(pNB-Est13 part1)=24 ng/µL
- c(pNB-Est13 part2)=26 ng/µL
- c(EstA part2)=13 ng/µL
- new SOE EstA mut PstI out lo is orderd from Sigma-Aldrich
- SOE PCR
- gene of interest: pNB-Est13 for SOE PCR with EstA and Promo-LacO-RBS-Phoa
- primers: SOE b1 up & SOE b1 lo
- template: pNB-Est13 part1 from last friday & pNB-Est13 part2
- annotation: TA1 = 52°C, ta1 = 25 s, tE1 = 35 s
- TA2 = 57°C, ta2 = 25 s, tE2 = 1.15 min
- Agarose gel electrophoresis
Tuesday, 15.05.12
- SOE PCR
- gene of interest: Promo-LacO-RBS-Phoa-pNBEst13 for SOE PCR with EstA
- primers: SOE A up & SOE b1 lo
- template: pNB-Est13 from yesterday & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12
- SOE PCR
- gene of interest: Promo-LacO-RBS-Phoa-FsC for SOE PCR with EstA
- primers: SOE A up & SOE b2 lo
- template: FsC from Thursday, 26.04.12 & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12
- annotation: TA1 = 52°C, ta1 = 25 s, tE1 = 35 s
- TA2 = 57°C, ta2 = 25 s, tE2 = 1.15 min
- each PCR is performed in 2 batches à 50 µL
Wednesday, 16.05.12
- SOE PCR of [[Promo-LacO-RBS-Phoa-pNBEst13] did not work but Promo-LacO-RBS-Phoa-FsC worked
- Gel extraction
- c(Promo-LacO-RBS-Phoa-FsC)=10 ng/µL
Week 5 / Kw 21
Monday, 23.05.12
- PCR on pEST100
- gene of interest: EstA part1 for SOE PCR with pNB-Est13 and EstA part2
- primers: SOE c1 up & SOE EstA mut PstI out lo
- PCR on pEST100
- gene of interest: EstA part1 for SOE PCR with FsC and EstA part2
- primers: SOE c2 up & SOE EstA mut PstI out lo
Tuesday, 22.05.12
- qualitative Agarose gel electrophoresis
- Gel extraction
- c(EstA part1 for SOE PCR with FsC)=4 ng/µL
- c(EstA part1 for SOE PCR with pNB-Est13)=7 ng/µL
Wednesday, 23.05.12
- SOE PCR
- gene of interest: EstA for SOE PCR with pNB-Est13
- primers: SOE c1 up & SOE D lo
- template: EstA part1 from yesterday & EstA part2 from Monday, 14.05.12
- SOE PCR
- gene of interest: EstA for SOE PCR with FsC
- primers: SOE c2 up & SOE D lo
- template: EstA part1 from yesterday & EstA part2 from Monday, 14.05.12
- annotation: TA1 = 52°C, ta1 = 25 s, tE1 =45 s
- TA2 = 65°C, ta2 = 25 s, tE2 = 90 s
- qualitative Agarose gel electrophoresis
Thursday, 24.05.12
- PCR on EstA for SOE PCR with pNB-Est13 from yesterday
- PCR on EstA for SOE PCR with FsC
- PCR on pEST100
- gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
- primers: SOE A up & SOE a1 lo
- PCR on pEST100
- gene of interest: FsC for SOE PCR with Promo-LacO-RBS-Phoa and EstA part1
- primers: SOE b2 up & SOE b2 lo
- annotation: TA = 60°C, ta = 25 s, tE =45 s
- each PCR is performed in 3 batches à 50 µL
- qualitative Agarose gel electrophoresis
- both EstAs worked
- Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13 worked
- Gel extraction
Week 6 / Kw 22
Tuesday, 29.05.12
- SOE PCR
- gene of interest: Promo-LacO-RBS-Phoa-Fsc-EstA
- primers: SOE A up & SOE D lo
- template: EstA from Thursday, 24.05.12 & Promo-LacO-RBS-Phoa-Fsc from Wednesday, 16.05.12
- SOE PCR
- gene of interest: Promo-LacO-RBS-Phoa-pNBEst13-EstA
- primers: SOE A up & SOE D lo
- template: EstA from Thursday, 24.05.12 & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12 & pNB-Est13 from Monday, 14.05.12
- SOE PCR
- gene of interest: Promo-LacO-RBS-Phoa-pNBEst13 for SOE PCR with EstA
- primers: SOE A up & SOE b1 lo
- template: Promo-LacO-RBS-Phoa from Wednesday, 02.05.12 & pNB-Est13 from Monday, 14.05.12
- annotation: each PCR is performed in 3 batches à 50 µL
- TA1 = 52°C, ta1 = 25 s, tE1 =45 s
- TA2 = 65°C, ta2 = 25 s, tE2 = 90 s
- Agarose gel electrophoresis
- Promo-LacO-RBS-Phoa-Fsc-EstA worked
- Promo-LacO-RBS-Phoa-pNBEst13 worked
Thursday, 29.05.12
- Gel extraction
- c(Promo-LacO-RBS-Phoa-Fsc-EstA)= 8 ng/µL
- c(Promo-LacO-RBS-Phoa-pNBEst13)= 13 ng/µL
SKV
Week 1 / CW 17
Tuesday, 24.04.12
- Production of electrocompetent cells DH5alpha and BL21
- Pouring of LB-Agar plates with ampecilin resistance (AMP)
- setting of DYT media
- Electroporation of BL21 with the following plasmids
Wednesday, 25.04.12
- Miniprep of the 3 overnigth Bl21 cultures and concentration meassurement via Nanodrop
Thursday, 26.04.12
Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE = 25 s
- PCR on pEST100
- gene of interest: Promo-LacO-RBS-Phoa for SKV
- primers: SKV a1 up XbaI & SKV a1 lo NdeI
- Agarose gel electrophoresis (Agarose gele elektrophoresis) for qualitiy control
120426 PCR 1-3 siehe Laborbuch 26.4.tif
Friday, 27.04.12
- clean up of 1. PCR on pEST100 from Thursday, 26.04.12 via Ammonium acetate - Ehtanol DNA precipitation, solved in 54 µl ddH2O
- restriction of cleaned up 1. PCR and pET16b with XbaI and NdeI over weekend at 37°C
Week 2 / CW 18
Monday, 30.04.12
- Agarose gel electrophoresis of DNA digestion from Friday, 27.04.12
120430 Testrestrikt.XbaINdeI.tif
- Only one single band on Agarose gel electrophoresis image
- For saveness two single digests are performed at 20 µL batches. One with XbaI and the other with NdeI. Incubation for 2 hours.
- Agarose gel electrophoresis of single digests
- Enzymes cut once each, so digest should have worked.
- Ligation of PhoA in cut (XbaI / NdeI) pET16b(+)
- Annotation: 30 µL batch, 1:5 ratio, Ligation at 4°C for 2 days till Wednesday, 02.05.12
Week 4/CW 20
Monday, 07.05.
- Electroporation of DH5alpha with 5 µl of the ligation which was performed 2 days before.
- clean up of the PCR-product PhoA Friday, 04.05.
- Restriction digest of half of the product with XbaI/NdeI for 1,5h at 37°C
- PhoA is cleaned up by Agarose gel electrophoresis
- concentration:7,36 ng/µl,10,6 ng/µl
- Ligation of PhoA x pET16b(+) for 2h, at the rate of (Vector/Insert) 1:5, 1:3
Annotation: If it does not say anything else, Ligation is always done in 20µl batches.
Tuesday, 08.05.
- Transformation of DH5alpha with PhoA x pET16b(+) was not successfull, so it was performed again Monday, 07.05.
- DNA digestion of pET16b(+)
- 100 µl are cut with XbaI/NdeI, incubaton-time: 1,5h at 37°C
- product is cleaned up by Ammonium acetate - Ehtanol DNA precipitation
- concentration: 85 ng/µl
Wednesday, 09.05.
- Colony-PCR
- gene of interest: PhoA
- primer:SKV a1 up XbaI, SKV a1 lo NdeI
- PhoA was not inserted, but 2 colonies were picked and inoculated in 5 ml LB Amp medium to look at it again due to a test-restriction.
Thursday, 10.05.
- Miniprep of PhoA x pET16b(+)
- Restriction digest of the Miniprep: PhoA x pET16b(+)
- enzymes: XbaI/NdeI, incubation: 1,5 h, 37°C
- digest shows that PhoA was not inserted
- Ligation is performed again Monday, 07.05.
- Amplification of the genes FSC, Est13 and EstA via PCR gg 1
- templates:
- primer:
Friday, 11.05.
- DNA digestion of pET16b(+) to produce template for a new ligation of PhoA
- concentration: 76,3 ng/µl
- enzymes: XbaI, NdeI
- incubation: for 2 days, 37°C
- LIgation of PhoA x pET16b(+) cut with XbaI and NdeI is carried out using different rates of Vector and insert:
- ratio vector/insert= 3:1, 1:1, 1:3, 1:5, 1:7, 1:10
- PhoA= 10,6 ng/µl Monday,07.05.
- incubation: 2 days, 4°C
Week5/CW21
Monday, 14.05.
- the Restriction digest of pET16b(+) cut with XbaI and NdeI is isolated by Ammonium acetate - Ehtanol DNA precipitation
- LIgation of phoA x pET16b(+) is used to transform DH5alpha via Electroporation
Tuesday, 15.05.
- Colony-PCR
- primer:
- SKV a1 up XbaI,
- SKV a1 lo NdeI
-
- PhoA is inserted, so 10 clones are picked to grow them in 5ml DYT over night.
Wednesday, 16.05.
- Miniprep of 10 clones, the plasmid-DNA is isolated out of 3ml culture per clone.
- Restriction digest with NdeI and NcoI of the plasmids PhoA x pET16b(+) 1-4, so FSC and Est13 can be added to the construct.
- FSC is cut with NdeI and NcoI
- digests are performed in 20µl total volume at 37°C for 1,5h and cleaned up by Agarose gel electrophoresis
-
- PCR1 of EST13
- template: product of SOE-PCR, cleaned up by the Promega-Kit
- primer: SKV b1 up NdeI, SKV b1 lo NcoI
Friday, 18.05.
- Ligation of FSC x (phoA x pET16b(+))
- ratio vector/insert: 3:1, 1:1, 1:3
- concentration:
- (phoA x pET16b(+))= 3,72 ng/µl
- FSC= 12,22 ng/µl
Week 6/CW 22
Monday, 21.05.
- Elektroporation of DH5alpha with the Ligation of FSC x (phoA x pET16b(+))
- Restriction digest of (phoA x pET16b(+))(150µl,160µl) for the insertion of Est13/FSC and Est13(50µl)
- enzymes: NdeI, NcoI
- incubation: 1,5h, 37°C
- cleaned up viaAgarose gel electrophoresis
- c(phoA x pET16b(+))= 23,5 ng/µl
- c(Est13= 7,7 ng/µ
Tuesday, 22.05.
- Ligation of Est13 and (phoA x pET16b(+))
- both components have been cut with NdeI and NcoI Monday, 21.05.
- ratio vector/insert: 1:10, 1:5, 1:3
- incubation: 2h, 25°C
- Electroporation of dH5alpha using the Ligation of Est13 x (phoA x pET16b(+), 5 µl per 100µl cells
Wednesday, 23.05.
- colony-PCR
- aim is to check the insertion of Est13
- Primer:SKV b1 up NdeI, SKV b1 lo NcoI
- 8 colonies have been picked and the Agarose gel electrophoresis proves, that Est 13 has not been inserted into (phoA x pET16b(+)
Wednesday, 23.05.
- Ligation of FSC and Est13 in (phoA x pET16b(+)
- ratio vector/insert: 1:3, 1:5
- c(templates):Monday, 21.05.
- Elektroporation of DH5alpha with Est13 in (phoA x pET16b(+)
Thursday, 24.05.
- colony-PCR proves, that Est13 has been inserted into (phoA x pET16b(+)
- plates are covered with colonies, 8 colonies are chosen for colony-PCR
- colony No.5 is chosen to be inoculated in 5µ DYT/Amp medium
- [PCR 1] is performed again to amplify FSC and Est13
- products are cleaned up by Agarose gel electrophoresis
-
Friday, 25.05.
- Restriction digest of FSC and Est13 Thursday, 24.05.
- enzymes: NdeI, NcoI
- incubation: 2days, 37°C
week 7/CW 23
Tuesday, 29.05.
- Restriction digest of FSC and Est13 is cleaned up via Agarose gel electrophoresis
- MIniprep of Est13 x (phoA x pET16b(+) via Promega-Kit
- Ligation of FSC x (phoA x pET16b(+))
- ratio vector/insert: 1:3, 1:5, 1:10
- incubation: 2h, 25°C
- Electroporation of DH5alpha with 5µl of the LIgaton: FSC x (phoA x pET16b(+)
- PCR 1 to amplify EstA, (Thursday, 10.05.)
- 4 assays à 50 µL, TA = 66°C, ta = 35s, tE = 90s
- cleaned up via Kit
Wednesday, 30.05.
- Restriction digest of EstA (product of PCR, 80 µl) and Plasmid: (Est13 x phoA x pET16b(+))
- Electroporation of FSC x (phoA x pET16b(+) worked well, plates are covered with colonies which are picked to be grown in 5ml DYT/Amp medium over night.
Thursday, 31.05.
- Miniprep of the amplified (FSC x phoA x pET16b(+))
- Restriction digest to ligate EstA
- enzymes:NcoI, EcoRI
- incubation: 1,5h, 37°C
- to be sure that the plasmid (Est13 x phoA x pET16b(+)) really carries Est13 and PhoA, an analytic PCR1 is performed. Also pEt16b(+) is used as template with different primers.
- templates:
- primers, used on each template:
- conditions:
- TA = 66°C, ta = 35s, tE = 90s
- the Agarose gel electrophoresis shows, that there is no binding-specifity concerning the genes of interest and the primers, but primers have been checked on mistakes.
- to solve the problem, the LIgation of FSC and Est13 shall be repeated from the beginning.
- Image
- Additionally,the decision was to go on with the Ligation of EstA into the Plasmids (Est13 x phoA x pET16b(+)) and (FSC x phoA x pET16b(+)), in case that the PCR was proceeded under wrong conditions.
Friday, 01.06.
- Restriction digest of (phoA x pET16b(+))
- enzymes: NcoI, NdeI
- incubation: 1,5 h, 37°C
- LIgation of Est13 and FSCx(PhoAxpET16b(+))
- Restriction digest of (FSCx (PhoA x pET16b(+))
- enzymes: NcoI, EcoRI
- incubation: 1,5h, 37°C
week 8/CW 24
Monday, 04.06.
- restriction of (FSCx(PhoAxpET16b(+)) and (phoAxpET16b(+)) is cleaned up by Agarose gel electrophoresis
- Electroporation of DH5alpha with Ligation: (FSCxPhoAxpET16b(+)), (EST13xPhoAxpET16b(+)) Friday, 01.06
Tuesday, 05.06.
- colony-PCR on EST13 & FSC is negative.
Wednesday, 06.06.
- Electroporation of DH5alpha with Ligation-assays from 01.06.
- Ligation of EstAWednesday,30.05 and the Plasmids (Est13xphoAxpET16b(+)),(FSCxphoAxpET16b(+)) from Monday,04.06.
Friday, 08.06.
- No cells are grown on plates, so the plates are incubated for another two days at RT.
week8/ CW24
Monday, 11.06.
- all plates are covered with cells, so a colony-PCR is performed on either Est13,FSC or EstA
- primer:
- SKV b1 up NdeI, SKV b1 lo NcoI
- SKV b2 up NdeI, SKV b2 lo NCOI
- SKV c1 up NcoI, SKV c1 lo EcoRI
- Every PCR is done in 8 assays à 50 µL, TA = 57°C, ta = 35s, tE = 90 s
Tuesday, 12.06.
- Agarose gel electrophoresis shows, that EstA has been inserted in both Plasmids, (Est13xPhoAxpET16b(+)) and (FSCxPhoAxpET16b(+))
-
- colonies No.3,4 (EstAxFSCxPhoAxpET16b(+)) and No.15,16 (EstAxEst13xPhoAxpET16b(+)) seem to be positive on EstA and are picked to be grown in 5ml DYT/Amp medium.
- colony-PCR on Est13 and FSC shows a lot of unspecific bands, so cloning of the genes from the beginning has to be dropped.
Wednesday, 13.06.
- Miniprep of the clones No. 3,4,15 and 16. Tuesday, 12.06.
- for gene-expression, it is performed a Elektroporation of BL21-cells with 1µl of the PLasmid-DNA which has just been isolated.
Friday, 15.06.
- Transformation of BL21 worked well
- Tributyrinagar plates with 1mM IPTG were generated, to induce the Lac-Promotor of pET16b(+), and to demonstrate the esterase-activity by the lysis of Tributyrin.
- afterwards Tributyrinagar plates are inoculated with cells which were transformed by Miniprep No.3,4,15,16 on Wednesday, 13.06.
- incubation: 37°C, 2 days
week9/CW25
Monday, 18.06.
- Tributyrinagar plates are covered with cells, but no lysis is visible.
- plates are incubated for another day at 25°C.
Tuesday, 19.06.
- no lysis, plates stay incubated at 25°C.
Wednesday, 20.06.
- no lysis
Friday, 22.06.
- The plasmids No.3,4 (EstAxFSCxPhoAxpET16b(+)) and No.15,16 (EstAxEst13xPhoAxpET16b(+)) are prepared for sequencing at Seqlab
- primer:
- (EstAxFSCxPhoAxpET16b(+)):
- (EstAxEst13xPhoAxpET16b(+)):
Sequencing was not successfull. It was discovered later, that there was a contamination of the DH5alpha cells with other plasmid-DNA, probably caused by Electroporation. Verlinkung Arne Due to this fact, the isolated plasmids were always mixed up with other plasmids, and sequencing had to fail. The contamination of the cells may have also caused false-positive results of the colony-PCRs.
We suggest that the cutinase FSC has an additional lipase-activity, which causes a lysis of the cell-membrane and may have an selective effect on FSC-negative mutants while cultivating the cells.
BioBricks
Week 1 / Kw 29
Tuesday, 17.07.12
- each PCR is performed in 5 batches à 50 µL.
- TA = 57°C, tA = 30 s, tE = 2 min
- PCR on pEST100
- gene of interest: PhoA for designing part BBa_K808028
- primers: BBa PhoA up & BBa PhoA down
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part1 for designing part BBa_K808026 via SOE PCR
- primers: BBa Est13 up & SOE Est13 mut lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part2 for designing part BBa_K808026 via SOE PCR
- primers: BBa Est13 down & SOE Est13 mut up
- Agarose gel electrophoresis for quality control
- preparative Agarose gel electrophoresis
- Gel extraction of PCR products
- 1. PCR (gene of interest: phoA): c(PhoA)=19 ng/µL
- 2. PCR (gene of interest: pNB-Est13 part1): c(pNB-Est13 part1)=39 ng/µL
- 3. PCR (gene of interest: pNB-Est13 part2): c(pNB-Est13 part2)=40 ng/µL
- SOE PCR
- gene of interest: pNB-Est13 for designing part BBa_K808028
- primers: BBa Est13 up & BBa Est13 down
- Annotation: SOE PCR is performed in 5 batches à 50 µL
- TA1 = 52°C, tA1 = 25 s, tE1 = 75 s
- TA2 = 57°C, tA2 = 30 s, tE2 = 2 min
- stored over night at 10°C
Wednesday, 18.07.12
- qualitative Agarose gel electrophoresis of SOE PCR (from Tuesday, 17.07.12)
- preperative Agarose gel electrophoresis
- Gel extraction of SOE PCR produkt
- PCR 1
- gene of interest: EstA from SKV Date for designing part BBa_K808027
- primers: BBa EstA up and BBa EstA down
- Annotation: PCR is performed in 5 batches à 50 µL
- TA = 57°C, tA = 25 s, tE = 75 s
- GC Buffer is used
- preparative Agarose gel electrophoresis
- Gel extraction of PCR product
- gene of interest: EstA: c(pNB-Est13 part2)=119 ng/µL
- stored in freezer
Thursday, 19.07.12
- inoculation of 5 mL DYT-media with 5 µL CAM and DH5alpha carrying pSB1C3 with part BBa_K808025 (FsC)
- DNA Digestion of the following parts
- PhoA from Tuesday, 17.07.12
- pNB-Est13 fromTuesday, 17.07.12
- EstA from Wednesday, 18.07.12
- pSB1C31
- pSB1C32
- adding of Dephosphatase Antarctica from NEB on digested pSB1C3 1 & 2
- Ligation into digested and 5' dephosphorylated pSB1C3 1
- bacterial transformation by Electroporation of DH5alpha
Friday, 20.07.12
- the following Electroporation from Thursday, 19.07.12 worked
- inoculation of 5 mL DYT-media with 5 µL CAM and two positive colonies from each transformation (from pNB-Est13 3 colonies are taken) .
- in addition FsC is already on plate, but to controle it, 2 colony PCRs are performed
- Ligation of EstA into digested and 5' dephosphorylated pSB1C3
- Electroporation of DH5alpha
- Miniprep of inoculated FsC in pSB1C3 (BBa_K808025) from Thursday, 19.07.12 failed
- inoculation of 5 mL DYT-media with 5 µL CAM and DH5alpha carrying pSB1C3 with part BBa_K808025 (FsC)
- incubation at 37°C over weekend of transformations and picked colonies
Week 2 / CW 30
Monday, 23.07.12
- Colony PCR of picked colonies from Friday, 20.07.12
120723 BBaPhoA BBa PhoA BBa FsC BBa FsC BBa Est13 BBa Est13 BBa Est13.jpg
- 2-3 PhoA colonies, 4-5 FsC colonies, 6-8 pNB-Est13 colonies
- Miniprep of one inoculated colony in DYT-media with CAM from Friday, 20.07.12
- inoculation of 2 x 5 mL DYT-media-CAM with pSB1C3 carrying FsC
- second transformation of EstA from Friday, 20.07.12 worked, but too many colonies!
- purity plate is done
Tuesday, 24.07.12
- Miniprep of the 2 FsC DYT-media-CAM cultures with pSB1C3 carrying FsC from yesterday
- purity plate of EstA in pSB1c3 is positive
- colonies picked
- Colony PCR with positive control by amplfifying an SKV-EstA
- Annotation: TA = 57°C, tA = 25 s, tE = 2 min, done with NEB- Phusion so its similiar to PCR 1
- GC Buffer is used
Wednesday, 25.07.12
- Agarose gel electrophoresis of Colony PCR
- worked out well
120725 Colony BBaEstA positiv probe auf SKV EstA.jpg
Thursday, 26.07.12
- Sequencing is ordered. The following premixes are used :
- 10 µL of c(PhoA in pSB1C3)=220 ng/µL, 1µL VR, 9 µL ddH2O
- 10 µL of c(PhoA in pSB1C3)=220 ng/µL, 1µL VF2, 9 µL ddH2O
- 7 µL of c(FsC in pSB1C3)=168 ng/µL, 1µL VR, 7 µL ddH2O
- 7 µL of c(FsC in pSB1C3)=168 ng/µL, 1µL VF2, 7 µL ddH2O
- 5 µL of c(EstA in pSB1C3)=350 ng/µL, 1µL VR, 9 µL ddH2O
- 5 µL of c(EstA in pSB1C3)=350 ng/µL, 1µL VF2, 9 µL ddH2O
- 5 µL of c(pNB-Est13 in pSB1C3)=50 ng/µL, 1µL VR, 9 µL ddH2O
- 5 µL of c(pNB-Est13 in pSB1C3)=50 ng/µL, 1µL VF2, 9 µL ddH2O
- in order to prepare DMSO stocks and Minipreps 5 mL DYT-media-CAM are inoculated with following colonies containing
Friday, 27.07.12
- inoculated pNB-Est13 in pSB1C3 from Thursday, 26.07.12 has not grown over night
- new colony pcked from plate and incubated in 5 mL DYT-media-CAM at 37°C over night
- Miniprep of 3 mL from the following over night DYT-media cultures from Thursday, 26.07.12
Week 3 / CW 31
Monday, 30.07.12
- bacterial transformation by Electroporation of DH5alpha with c(pNB-Est13 in pSB1C3)=50 ng/µL from Monday, 23.07.12
Tuesday, 31.07.12
- no results in sequencing our BioBricks / parts from Thursday, 26.07.12 except for FsC in pSB1C3
- new Colony PCR(similar to PCR 2) is done on
- no signal on qualitative Agarose gel electrophoresis
- Trouble shooting (see discussion below)
Trouble shooting
- Symptoms
- very much transformants on crossed out plates after bacterial transformation by Electroporation
- at first Colony PCR is positive
- Minipreps result in very high yields (exceeding >200 ng/µL)
- second Colony PCR is negative
- sequencing failed
- BUT FsC in pSB1C3
- has had not very much transformants
- has had a seuqencing result
- its Minipreps resulted in medium concentrations, settling around 90 ng/µL
- Diagnosis:
- our bacterial transformation is done by Electroporation
- therefor we use electroporation cuvettes, stored in DNA precipitating liquids like isopropanol or ethanol after being washed out.
- long before we started these electroporation cuvettes were in use.
- if not washed out carefully, DNA debris (like vectors and Inserts) accumulates in these storage liquids, due to precipitation
- when an Electroporation is performed with one of these cuvettes, probably containing only little DNA debris, the cells get transformed with numbers of different precipitated plasmids
- these plasmids contain a variety of resistances, reaching from CAM over KAN to Amp, allowing even "wrong" transformants to grow on selective media
- after a short period of time, bacterial colonies start to seperate and repel their plasmids, selecting only the most "comfortables"
- probably our plasmids, containing big genes on ligated vectors, are pushed out during this phase of selection
- this slow decrease of plasmid could explain the missing second positive signal of Colony PCR
- Strategy
- switching our method from Electroporation to Heatshock transformation
Wednesday, 01.08.12
- due to change of strategy the following PCR 1s are performed
- PCR on pEST100
- gene of interest: PhoA BioBrick for assembly of part BBa_K808028
- primers: BBa PhoA up & BBa PhoA down
- PCR on pEST100
- gene of interest: EstA part1 for SOE PCR of EstA (BBa_K808027)
- primers: BBa Est A up & SOE EstA mut PstI out lo
- PCR on pEST100
- gene of interest: EstA part2 for SOE PCR of EstA (BBa_K808027)
- primers: BBa EstA mut up & BBa EstA down
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part1 for SOE PCR of pNB-Est13 (BBa_K808026)
- primers: BBa Est13 up & BBa Est13 mut lo
- PCR on pET26b(+)
- gene of interest: pNB-Est13 part2 for SOE PCR of pNB-Est13 (BBa_K808026)
- primers: BBa Est13 lo & BBa Est13 mut up
- Annotation: every PCR is performed in 3 batches à 50 µL
- TA = 57°C, tA = 25 s, tE = 75 s
- due to time pressure there will be no qualitative but only preperative Agarose gel electrophoresis
- PhoA only on analytical scale but on same Agarose gel electrophoresis with EstA part1
120801 soe pcr esta1 pcr phoa.jpg
- 1 cut out EstA part1, 2 PhoA
120801 soe pcr est131 est132.jpg
- 2 cut out pNB-Est13 part1, 3 cut out pNB-Est13 part2
- cut out EstA part2
- Gel extraction of EstA part1, EstA part2, pNB-Est13 part1 and pNB-Est13 part2
- c(EstA part1)=19 ng/µL
- c(EstA part2)=110 ng/µL
- c(pNB-Est13 part1)=45 ng/µL
- c(pNB-Est13 part2)=48 ng/µL
- SOE PCRs
- SOE PCR
- gene of interest: EstA
- primers: EstA part1 & EstA part2
- SOE PCR
- gene of interest: pNB-Est13
- primers: pNB-Est13 part1 & pNB-Est13 part2
- as a control a PCR is performed on EstA part2
- Annotation: SOE PCR is performed in 3 batches à 50 µL
- TA1 = 52°C, tA1 = 30 s, tE1 = 75 s
- TA2 = 57°C, tA2 = 30 s, tE2 = 75 s
- stored over night at 10°C
- qualitative Agarose gel electrophoresis
120801 SOE PCR Est13 EstA EstApart2 kontrolle.jpg
- 2 pNB-Est13, 3 ESTA, 4EstA part2
- PCR Clean up by Promega Kit of PhoA, pNB-Est13, EstA
- DNA Digestion of the following parts
Thursday, 02.08.12
- Ligation into digested and 5' dephosphorylated pSB1C3
- pNB-Est13
- PhoA
- EstA
- Annotation: Ligation is performed in a 20 µL batch
- digested pSB1C3 still availabe from Thursday, 19.07.12
- 1:5 & 1:10 ratio
- incubation at room temperature for 30 min
- Heatschock transformation of DH5alpha with our ligation batches
- incubation over night at 37°C
Friday, 03.08.12
- transformation succeeded
- colonies picked to inoculate 5 ml DYT-media-CM
Saturday, 04.08.12
- Miniprep of picked colonies
- test DNA Digestion with EcoRI-HF and PstI-HF
- PCR 2 on preped plasmids with primers VR & VF2, and the respective BBa primers
- TA = 60°C, ta = 35s, tE = 25 s
- qualitative Agarose gel electrophoresis 1% agarose
120804 Verdau PhoA, EstA, Est13, PCR auf EstA, Est 13, PhoA, Kontrolle auf Insert und Vektor.jpg
- digestion: 2 PhoA, 3 EstA, 4 pNB-Est13 PCR: 6 EstA with BBa primers, 7 EstA with VR & VF2, 8 pNB-Est13 with BBa primers, 10 pNB-Est13 with VR & VF2, 11 PhoA with BBa primers, 12 PhoA with VR & VF2
- tests succeeded
- Annotation: failure in mixing sequencing premixes
- retransformation of DH5alpha by Heatshock transformation with the following plasmids with 1 µL each
Week 4 / Kw 32
Assembly of our composite part RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808030) is reached by synthesis (from GENEART) of 2 different gene parts:
- bba prefix-PhoA-His6tag-pNBEst13-BsaI site
- BsaI-Myctag-EstA- bba suffix
- the RBS will be added by PCR with primers XbaI Rbs Phoa up (ignoring the bba prefix but adding an XbaI site upstream of the RBS) & Est13 Bsa1 lo, ignoring the bba prefix but adding an XbaI site upstream of the RBS and for the downstream part primers EstA Bsa1 up andBBa EstA down are used
- both products can be assembled by using BsaI. An restriction enzyme, with an restriction site in a defined distance to its recognition site
Monday, 06.08.12
- Heatshock transformation of DH5alpha with 1 µL synthesis products (c=100 ng/µL):
- DNA Digestion of 10 µL = 1 µg of both synthesis products
- bba prefix-PhoA-His6tag-pNBEst13-BsaI site digested with EcoRI-HF and BsaI-HF in a 50 µL batch
- BsaI-Myctag-EstA- bba suffix digested with BsaI-HF and PstI-HF in a 50 µL batch
- preparative Agarose gel electrophoresis
- Failure due to overlooked additional PstI site in GENEART Vectors
- inoculation of 5 ml DYT-media-CAM with retransformations from Saturday, 04.08.12
- incubation at 37°C over night
Tuesday, 07.08.12
- Colony PCR on 4 colonies of each transformation of synthesis product from Monday, 06.08.12
- Annotation: PCR is done with house-taq, similar to PCR 2
- TA = 57°C, tA = 30 s, tE = 90 s
- Colony PCR succeeded
- from bba prefix-PhoA-His6tag-pNBEst13-BsaI site colonies 5, 2, 11 are picked
- from BsaI-Myctag-EstA- bba suffix colonies 1, 10, 16 are picked
- inoculation of 5 ml DYT-media-KAN with colonies
- incubation over night at 37°C
- inoculated 5 mL cultures from Monday, 06.08.12 are stored at 4°C
Wednesday, 08.08.12
- Miniprep of following 5 mL cultures
- from Monday, 06.08.12
- from Tuesday, 07.08.12
- bba prefix-PhoA-His6tag-pNBEst13-BsaI site
- colony 2 c=438 ng/µL
- colony 5 c=509 ng/µL
- colony 11 c=291 ng/µL
- BsaI-Myctag-EstA- bba suffix
- colony 1 c=381 ng/µL
- colony 10 c=420 ng/µL
- colony 16 c=263 ng/µL
- bba prefix-PhoA-His6tag-pNBEst13-BsaI site
- DNA Digestion
- of bba prefix-PhoA-His6tag-pNBEst13-BsaI site colony 11
- performed in a 40 µL batch
- BsaI and EcoRI are used
- digestion for 3 h
- of BsaI-Myctag-EstA- bba suffix
- performed in a 40 µL batch
- BsaI and SpeI are used
- digestion for 3 h
- of bba prefix-PhoA-His6tag-pNBEst13-BsaI site colony 11
- preperative Agarose gel electrophoresis
120808 Restriktion mit E BsaI PhoA Est13 und bsai s EstA.jpg
- 3-4 cut out bba prefix-PhoA-His6tag-pNBEst13-BsaI site, 5-6 cut out BsaI-Myctag-EstA- bba suffix
- Gel extraction
- c(bba prefix-PhoA-His6tag-pNBEst13-BsaI site)= 8 ng/µL
- c(BsaI-Myctag-EstA- bba suffix)= 26 ng/µL
- ligation of both inserts in pSB1C3 2 from Thursday, 19.07.12
- 1:5:5 ratio is performed in two 20 µL batches (one incubated at 4°C, the other at room temperature over night)
- new sequencing is ordered of the following plasmids
Thursday, 09.08.12
- Heatshock transformation of DH5alpha with 10 µL of each ligation batch from yesterday
Friday, 10.08.12
- transformation from yesterday suceeded
- one colony per plate (1:5:5 & 1:10:10 at room temperature and 1:5:5 & 1:10:10 at room 4°C)is picked for inoculation of 5 mL DYT-media-CAM
Saturday, 11.08.12
- Miniprep of picked colonies from yesterday
- testing ligation by DNA Digestion of plasmids with EcorI-HF & PstI-HF
- Colony PCR on picked colonies
- Ligation and transformation succeeded
120811 PCR (VR,VF2) und Verdau (EcoRI,PstI) auf BBa Gesamt von Geneart.jpg
- 2-5 bba prefix-PhoA-His6tag-pNBEst13-Myctag-EstA- bba suffix running at round about 3 kb
Week 5 / CW 33
Monday, 13.08.12
- Preparation for designing part BBa_K808032 which is an arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
- in order to add RBS upstream of PhoA of bba prefix-PhoA-His6tag-pNBEst13-BsaI site the following PCRs are performed
- PCR 1 on bba prefix-PhoA-His6tag-pNBEst13-BsaI site, as a template the pure synthesis product is used
- TA = 57°C, tA = 25 s, tE = 2 min
- primers: XbaI Rbs Phoa up & Est13 Bsa1 lo
- PCR 1 on BsaI-Myctag-EstA- bba suffix, as a template the pure synthesis product is used
- TA = 57°C, tA = 25 s, tE = 2 min
- primers: EstA Bsa1 lo & BBa EstA down
- PCR 3 on preped colony (1:5:5 room temperature from Friday, 10.08.12)
- TA = 57°C, tA = 25 s, tE = 2 min
- primers: XbaI Rbs Phoa up & BBa EstA down
- Annotation: each PCR is performed in 4 batches à 50 µL
- 2 qualitative Agarose gel electrophoresis
120813 PCR 1-4 RBSPE 5-8 EXN-Myc-Est 9-12.jpg
120813 PCR Q5 auf bba gesamt konstrukt.jpg
- 2-4 bba prefix-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA-bba suffix
- PCRs succeeded
- PCR clean up of 1.-3. PCR
- DNA Digestion of pSB1C3 carrying RFP part ????
- DNA Digestion of part BBa_K808000 (pSB1C3 carrying Arabinose inducible promotor)
- preparative Agarose gel electrophoresis
- c(1.PCR)= 147 ng/µL
- c(2.PCR)= 133 ng/µL
- c(3.PCR)= 96 ng/µL
- c(cut S/P BBa_K808000: Arabinose inducible promotor) possibility 1= 14 ng/µL
- c(cut S/P BBa_K808000: Arabinose inducible promotor) possibility 2= 14 ng/µL
- c(cut X/P pSB1C3 carrying RFP)=20 ng/µL
- Ligation at 4°C over night of the following combinations:
- 1.PCR & 2.PCR in pSB1C3 carrying RFP
- 3.PCR in pSB1C3 carrying RFP
- 1.PCR & 2. PCR in BBa_K808000 possibility 1
- 3.PCR in BBa_K808000 possibility 1
- 1.PCR & 2. PCR in BBa_K808000 possibility 2
- 3.PCR in BBa_K808000 possibility 2
Tuesday, 14.08.12
- Heatshock transformation of ligation batches 1-6 from yesterday
Wednesday, 15.08.12
- transformation succeeded
- inoculation of 5 mL DYT-media-CAM of the following colonies
- colony 1.1
- colony 2.1
- colony 2.2
- colony 3.1
- colony 4.1
- colony 4.2
- colony 5.1
- colony 5.2
- colony 6.1
- colony 6.2
- incubation over night at 37°C
- Colony PCR on colonies 1.1-6.2 with house-taq
Thursday 16.08.12
- Miniprep of colonies from yesterday
- DNA Digestion of preped colonies with EcorI-HF & PstI-HF
- incubation for 1.5 h at 37°C
- qualitative Agarose gel electrophoresis
- FAILURE, because bands on gel are inconsistent
120816 Testrestriktion der pSB1C3 mit e und p colony platten 1 - 6.jpg
- 2 colony 1.1, 3 colony 2.1, 3 colony 2.2, 4 colony 3.1, 5 colony 4.1, 6 colony 4.2, 7 colony 5.1, 8 colony 5.2
- for further information see discussion below
- new inoculation of 5 mL DYT-media-CAM of Colonies 1.1 - 5.2
Trouble shooting
What should have been transformed:
- colony 1.1: 1.PCR & 2.PCR in pSB1C3
- should be RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808030) in pSB1C3 (length: ~ 3.1 kb)
- colony 2.1: 3.PCR in pSB1C3 upstream of part BBa_K808000 (Arabinose inducible promotor) possibility 1
- should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808032) in pSB1C3 (length: ~ 4.3kb)
- colony 2.2: similar to colony 2.1
- colony 3.1: 3.PCR in pSB1C3 upstream of part BBa_K808000 (Arabinose inducible promotor) possibility 2
- should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808032) in pSB1C3 (length: ~ 4.3kb) like colony 2.1
- colony 4.1: 3.PCR in pSB1C3
- should be RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808030) in pSB1C3 (length: ~ 3.1 kb)
- colony 4.2: similiar to colony 4.1
- colony 5.1: 1.PCR & 2.PCR in pSB1C3 upstream of part BBa_K808000 (Arabinose inducible promotor) possibility 1
- should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808032) in pSB1C3 (length: ~ 4.3kb)
- colony 5.2: similar to colony 5.1
Conclusion:
- estimated gene length was aorund 4,3 kb (colony 5.1, 5.2, 2.1, 2.2 = part BBa_K808032) or 3,1 kb (colony 1.1, 4.1, 4.2 part BBa_K808030)
- digestion with EcoRI-HF & PstI-HF of all ligations from Thursday 16.08.12 in part BBa_K808000 (Arabinose inducible promotor) possibility 1 shows bands at round about 3 - 3.5 kb
- when digested with same enzymes of ligations into part BBa_K808000 (Arabinose inducible promotor) possibility 2, bands are shown at round about 1 - 1.5 kb
- this leads to our concluison that BBa_K808000 (Arabinose inducible promotor) possibility 1 was the not digested DNA band, because resulting gene length of cut insert correlates with length of BBa_K808000 (Arabinose inducible promotor)
- for a sharper solution an Agarose gel electrophoresis with 0.5 % agarose is performed for digestions of colonies 1.1, 3.1, 4.1, 4.2 plus batches from Colony PCR from Wednesday, 15.08.12
120816 Colony 1.1 3.1 4.1 4.2 Colony Pcr 1-8.tif
- 2-5 colonies 1.1, 3.1, 4.1, 4.2, 6 - 13 colony 1.1, 2.1, 2.2, 3.1, 4.1, 4.2, 5.1, 5.2
Friday, 17.08.12
- the image above means that:
- colony 5.1, 5.2, 2.1, 2.2 are only carrying part [[BBa_K808000 which encodes for the arabinosis inducable promotor
- but colony 1.1, 2.1, 2.2 are carrying 1.PCR & 2.PCR or 3.PCR in pSB1C3
- Miniprep of named colonies in liquid DYT-culture from Thursday 16.08.12
- half culture is preped, half culture serves for 10% DMSO stocks
- c(1.1: BBa_K808030)= 81 ng/µL
- c(4.1: BBa_K808030)= 128 ng/µL
- c(4.2: BBa_K808030)= 79 ng/µL
- c(2.1: BBa_K808000)= 90 ng/µL
- c(2.2: BBa_K808000)= 78 ng/µL
- c(5.1: BBa_K808000)= 64 ng/µL
- c(5.2: BBa_K808000)= 62 ng/µL
- for BioBrick assembly we perform a DNA Digestion of
- BBa_K808000 (colony 1.1, 4.1, 4.2), with EcoRI / SpeI, serves as upstream part
- BBa_K808030 (colony 5.1, 5.2, 2.1, 2.2), with EcoRI / XbaI, serves as downstream part
- preperative Agarose gel electrophoresis
- Gel extraction
- downstream part
- c(1.1: BBa_K808030)= 21.5 ng/µL
- c(4.1: BBa_K808030)= 54 ng/µL
- c(4.2: BBa_K808030)= 58 ng/µL
- upstream part
- c(2.1: BBa_K808000)= 12 ng/µL
- c(2.2: BBa_K808000)= 11 ng/µL
- c(5.1: BBa_K808000)= 10 ng/µL
- c(5.2: BBa_K808000)= 5 ng/µL
- ligation of upstream infron of downstream part
- colony 5.1 (BBa_K808000) in colony 1.1 (BBa_K808030)
- colony 2.2 (BBa_K808000) in colony 4.2 (BBa_K808030)
- colony 2.1 (BBa_K808000) in colony 4.1 (BBa_K808030)
- inoculation of 5 mL DYT-media-CAM of the following colonies
- colony 1.1
- colony 2.1
- colony 2.2
- colony 3.1
- colony 4.1
- colony 4.2
- colony 5.1
Week 6 / CW 34
Monday 20.08.12
- Miniprep of incoucaltes colonies from Friday, 17.08.12
- c(1.1: BBa_K808030)= 54 ng/µL
- c(4.1: BBa_K808030)= 74 ng/µL
- c(4.2: BBa_K808030)= 60 ng/µL
- c(2.1: BBa_K808000)= 47 ng/µL
- c(2.2: BBa_K808000)= 42 ng/µL
- c(5.1: BBa_K808000)= 56 ng/µL
- sequencing of
- colony 1.1 using VR & VF2, 1.5 µL each
- colony 5.1 using VR, SOE EstA mut lo, SOE EstA mut up & VF2, 1.5 µL each
Tuesday 21.08.12
- Ligation of BBa_K808000 upstream of BBa_K808032 in pSB1C3 ( to design BBa_K808032) from Friday, 17.08.12 worked well
- 6 colonies are picked in order to perform a Colony PCR and for inoculation of 5 mL DYT-media-CAM
- colony A 4.1/2.1 ligated at 4°C
- colony B 4.1/2.1 ligated at 37°C
- colony C 1.1/5.1 ligated at 4°C
- colony D 1.1/5.1 ligated at 37°c
- colony E 4.2/2.2 ligated at 4°C
- colony F 4.2/2.2 ligated at 37°C
- Colony PCR
- Agarose gel electrophoresis
- did not work due to immense gene legth ( around 4 kb)
- Restriction digest for testing is planned
- inoculation of 5 mL DYT-media-CAM with colony E,F,C,D
Wednesday, 22.08.12
- Miniprep of inoculated 5 mL DYT-media-CAM with colony E,F,C,D
- c(colony C 1.1/5.1 ligated at 4°C)=46 ng/µL
- c(colony D 1.1/5.1 ligated at 37°c)=46 ng/µL
- c(colony E 4.2/2.2 ligated at 4°C)=60 ng/µL
- c(colony F 4.2/2.2 ligated at 37°C)=58 ng/µL
- DNA Digestion of 15 µL of preped plasmids with EcoRI-HF & PstI-HF
- expected insert length: 4.4 kb (BBa_K808000+BBa_K808030)
- Agarose gel electrophoresis with 0.8% agarose
Verdau vom Gesamtkonstrukt 4.2_2.2 und 5.1_1.1 beide temperaturen.tif
- all bands are in same heights, so ligation worked well
- ligation worked well, bands are in estimated length
- due to rather bad results of Miniprep colonies are used for inoculation of 5 mL DYT-media-CAM again
- incubation over night at 37°C
- evaluation of sequencing from Wednesday, 08.08.12
- PhoA did not work
- EstA is sequenced inconsistent
- pNB-Est13 still got its illegal PstI-site
- but at leat FsC is sequenced completely
- new sequencing of
- PhoA in pSB1C3, primers VF2 & VR, 1.5 µL each
- EstA in pSB1C3, primers SOE EstA mut up & BBa EstA down, 1.5 µL each
- PCR 1 performed on BBa_K808032(preped colony C 1.1/5.1 ligated at 4°C)
- TA = 57°C, tA = 25 s, tE = 2 min
- primers BBa Est13 up & BBa Est13 down
- performed in 3 batches à 50 µL
- analytical Agarose gel electrophoresis 2% agarose
- [PCR Clean up]]
- DNA Digestion of PCR product with EcoRI-HF & PstI-HF over night at 37°C
Thursday, 23.09.12
- Miniprep of colonies from
- c(colony C 1.1/5.1 ligated at 4°C)=147 ng/µL
- c(colony D 1.1/5.1 ligated at 37°c)=131ng/µL
- c(colony E 4.2/2.2 ligated at 4°C)=85 ng/µL
- c(colony F 4.2/2.2 ligated at 37°C)=128 ng/µL
- preparative Agarose gel electrophoresis of digested pNB-Est13 from yesterday
- Gel extraction leads to c(pNB-Est13- cut E/P)=25 ng/µL
- Ligation of digested pNB-Est13 in digested pSB1C3
- 1:5 ratio is used
- Heatshock transformation of DH5alpha with 10 µL of ligation batch
- incubation of transformed cells at 37°C over night
Friday, 24.08.12
- transformation of ligation from yesterday succeeded, colony picked and inoculation of 5 mL DYT-media-CAM
- sequencing of
- BBa_K808032 (colony C 1.1/5.1 ligated at 4°C), primers VF2, XbaI Rbs Phoa up, BBa Ara Promo lo, SE Est13 mut up, SOE Est13 mut lo, BBa EstA down, VR, 1.5 µL each,
Week 7 / CW 35
Monday, 27.08.12
- Miniprep of picked colony from BBa_K808026 (pNB-Est13 in pSB1C3)
- sequencing of pNB-Est13 in pSB1C3
- using primers VF2, VR, SOE Est13 mut up, SOE Est13 mut lo
- test expression of BBa_K808032 (colony C 1.1/5.1 ligated at 4°C) in DH5alpha
- BBa_K808032 is an arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
- inoculation of 50 mL DYT-media-CAM with DH5alpha carrying BBa_K808032
- stop incubation at at OD600=0.7
- storaging cultures on ice for 15mins
- inducing with ~ 1.5% L-arabinose (using 3.75 mL of 20% mw L-arobinose solution)
- incubation at 20°C, 25°C and 30°C over night
Tuesday, 28.08.12
- antibody staining
- detection of surface expression of BBa_K808032 by using FACS
- positive signal increases with higher temperature, staining succeeded
- Protein ourification of test expression without rinnung IMAC afterwards, just using supernatant and cell debris pellet
- 32 µL of supernatant is used
- 3 mL pellet of each testexpression is treated with more SDS as a detergent, in order to solve membrane proteins
- running two SDS-tris gels with an myc positive probe
- one of these gels is used for performing a Westernblot
- using mouse-antimyc antibody as first antibody and goat-antimouse-AP as second antibody
120831 Westermblot auf Gesamt konstrukt aus DH5 alpha 5111.tif
- both are not very good in their solution, we do it again with Laemmli gels
Wednesday, 29.08.12
- evalutation of sequencing from last week:
- PhoA worked
- EstA worked
- pNB-Est13 worked
- RBS-PhoA-His6tag-pNBEst13-Myctag-EstA worked
- arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
Thursday, 30.08.12
- running 2 SDS-Laemmli gels
- pellet is treated with n-Dodecyl-ß-maltoside (DDM) in order to solve membrane proteins
- one gel is used to perform a second Western blot with a myc positive probe
120831 Westermblot 2 auf Gesamt konstrukt aus DH5 alpha 5111.tif
- 2 supernatant 30°C, 3 pellet (treated with detergent) 30°C, 4 supernatant 25°C, 5 pellet (treated with detergent) 25°C, 6 supernatant 20°C, 7 pellet (treated with detergent) 20°C, 8 myc positive probe
- as we can see, only the treated 120831 Westermblot 2 auf Gesamt konstrukt aus DH5 alpha 5111pellets show positive signals when treated with antibodies, so we have expressed our membrane protein BBa_K808032
- now our lab can start with quantification of enzymatic activites of BBa_K808032 on PET or model substrates
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