Team:TU Darmstadt/Labjournal/Degradation

From 2012.igem.org

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(Procedure)
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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.
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.
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== Activity tests of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] ==
 
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=== Week 1 / CW 35 ===
 
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==== Friday, 31.08.12 ====
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5α] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] (arabinose inducable RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
 
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{| class="wikitable"
 
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|-
 
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! Component !! test tube 1 !! test tube 2 !! test tube 3 !! test tube 4
 
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|-
 
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| DYT-medium || 8 mL || 8 mL || 8 mL || 8 mL
 
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|-
 
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| PET particle || yes || yes || yes || yes
 
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|-
 
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| bacteria || yes || yes || yes || no
 
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|-
 
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| induced || 1.5% L-arabinose || 1.5% L-arabinose || no || no
 
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|}
 
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* test seemed to have worked: but an induced test tube without PET-granula was missing
 
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=== Week 2 / CW 36 ===
 
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==== Tuesday, 04.09.12 ====
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5α] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 
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{| class="wikitable"
 
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|-
 
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! Component !! test tube 1 !! test tube 2 !! test tube 3 !! test tube 4 !! test tube 5
 
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|-
 
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| DYT-medium || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL
 
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|-
 
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| PET particle || yes || yes || no || yes || yes
 
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|-
 
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| bacteria || yes || yes || yes || no || yes
 
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|-
 
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| induced || 1.5% L-arabinose || 1.5% L-arabinose || 1.5% L-arabinose || no || no
 
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|}
 
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*  test tube 3: [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] without bacteria contains [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Kanamycin Kan], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Ampicillin AMP]
 
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* looks good but test tube 2 shows no significant change of colour
 
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==== Wednesday, 05.09.12 ====
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5α] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 
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{| class="wikitable"
 
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|-
 
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! Component !! tube 1 !! tube 2 !! tube 3 !! tube 4 !! tube 5 !! tube 6 !! tube 7 !! tube 8 !! tube 9
 
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|-
 
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| DYT-medium || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL || 8 mL
 
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|-
 
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| PET particle || yes || yes || yes || yes || no || no || yes || yes || yes
 
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|-
 
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| bacteria || yes || yes || yes || yes || yes || yes || yes || yes || no
 
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|-
 
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| 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
 
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|}
 
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* test tube 9: [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] without bacteria contains [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Kanamycin Kan], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Ampicillin AMP]
 
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* all induced tubes turned yellow, even without PET-granula as a substrate
 
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* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/GFP GFP]
 
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==== Trouble shooting ====
 
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* 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%)
 
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* 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]
 
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* 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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==== Thursday, 06.09.12 ====
 
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*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5α] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 [BBa_K808032]] (arabinose inducable RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
 
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{| class="wikitable"
 
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|-
 
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! Component !! tube 1 !! tube 2 !! tube 3 !! tube 4 !! tube 5 !! tube 6 !! tube 7 !! tube 8 !! tube 9 !! tube 10 !! tube 11
 
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|-
 
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| DYT-medium || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL
 
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|-
 
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| PET particle || yes || yes || yes || yes || yes || yes || no || no || no || no || no
 
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|-
 
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| bacteria || yes || yes || yes || yes || yes || yes || yes || yes || yes || yes || no
 
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|-
 
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| 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
 
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|}
 
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* test tube 11: [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] without bacteria contains [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Kanamycin Kan], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Ampicillin AMP]
 
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* all induced tubes turned yellow, even without PET-granula as a substrate
 
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=== Week 3 / CW 37 ===
 
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==== Monday, 10.09.12 ====
 
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* we stopped the trial of quantification via PET-substrates because in terms of color the change of pH value (due to acid groups that are released by degradtion) is not very exactly. In order to get reproducible data, we are using new activity assays.
 
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* next bacterial assays are going to be in 96 well plates using a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#pNP-Assay pNP-Assay]
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Heat_Shock_Transformation Heatshock transformation] of [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay#LB-Agar-Tributyrin activity assay on LB-Tributyrin-CAM-plates] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose L-arabinose] concentrations: 0.1%, 0.2%, 0.4%
 
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** colony 1-5 are [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5 alpha] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] but colony 6 is just DH5 alpha carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 
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** incubated over night at 37°C
 
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==== Tuesday, 11.09.12 ====
 
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* inoculation of 5 mL [[DYT-media]]-CAM with K1 and K2 of transformed [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] from yesterday
 
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* acticity tests on [[LB-Tributyrin-CAM-plates]] shows good results
 
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** from now on incubation at room temperature
 
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[[File: 120911 dh5alpha k808032 0.1 ara.jpg|200px]]
 
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[[File: 120911 dh5alpha k808032 0.2 ara.jpg|200px]]
 
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[[File:120911 dh5alpha k808032 0.4 ara.jpg|200px]]
 
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==== Wednesday, 12.09.12 ====
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep#Promega_kit Plasmid preparation] of inoculated [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] colonies K1 and K2
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest DNA digestion] with EcoRI-HF & PstI-HF
 
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* qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis AGE]
 
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[[File:120912 Testrestriktion K808032 im ethylenglycol stamm K1 und K2 mit ecori und psti.tif]]
 
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==== Thursday, 13.09.12 ====
 
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* the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay#LB-Agar-Tributyrin activity assay on LB-Tributyrin-CAM-plates] from tuesday gives good results
 
-
 
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[[File:120913 dh5alpha k808032 0.1 ara.jpg|200px]] [[File:120913 dh5alpha k808032 0.2 ara.jpg|200px]] [[File:120913 dh5alpha k808032 0.4 ara.jpg|200px]]
 
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* to perform a better expression of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030] we will use [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] as a host for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]. Its unability to metabolize L-arabinose is crucial for our decision. We hope to generate good expression levels.
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] (after washing ecletroporation cuvettes very carefully) of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 
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==== Friday, 14.09.12 ====
 
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* yesterdays electroporation worked well
 
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* inoculation of 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT-media]-CAM with colony from plate of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 
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==== Saturday, 15.09.12 ====
 
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* making [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Glycerine_stock#Protocol 10% DMSO stocks] from 50 mL [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] culture
 
-
 
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=== Week 4/ KW 38 ===
 
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==== Monday, 17.09.12 ====
 
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* inoculation of 4 x 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT-media]-CAM with [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] stocks
 
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* at OD<sub>600</sub>=0,5 the cultures are incduced with:
 
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** 0.02% mw L-arabinose
 
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** 0.2% mw L-arabinose
 
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** 0.5% mw L-arabinose
 
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** one culture is not induced and will serve as a negative control
 
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* after 3 hours an [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Antibody_staining antibody staining] is performed on our expressed [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] containing a myc-epitope
 
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* checking surface expression levels via [http://en.wikipedia.org/wiki/Flow_cytometry#Labels flow cytometry]
 
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** inducing worked well, we can go on with screening the enzymatic activity
 
-
 
-
[[Image:Flow cytometry 002 ara.png|200px]] [[Image:Flow cytometry 02 ara.png|200px]][[Image:Flow cytometry 05 ara.png|200px]] [[Image:Flow cytometry alle.png|200px]]
 
-
 
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==== Tuesday, 18.09.12 ====
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030], serving as a negative  (directly used after 1h of incubation at 37°C in pure DYT-medium)
 
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* activity assay of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar] with L-arabinose concentrations: 0.02%, 0.2%, 0.5%
 
-
** colonies 1-3 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] but colony 4 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification#Procedure Protein purification] from induced cultures and negative control
 
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** cell debris pellets are treated with [http://www.sigmaaldrich.com/catalog/product/sigma/d4641?lang=en&region=US n-Dodecyl ß-D-maltoside] as a detergent
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page#SDS-PAGE_.28Laemmli.29 SDS PAGE (Laemmli)] of pellets and supernatants
 
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[[Image: SDS Page of BBa_K808030.png|400px|thumb|left|2: supernatant 0.5% arabinose, 3: treated pellet 0.5% arabinose, 4: supernatant 0.2% arabinose, 5: treated pellet 0.2% arabinose, 7: supernatant 0.02% arabinose, 8: treated pellet 0.02% arabinose, 9: supernatant no arabinose, 10: treated pellet no arabinose ]]
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Western_Blot Westernblot] is successful as well
 
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* to quantify the hydrolytic activity of our induced bacteria we perform a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#Bacteria bacterial pNP-Assay]
 
-
 
-
[[File:top_ten_induz(geschwindigkeiten)(1).png|500px|center|thumb|graph shows absorbtion, ergo hydrolytic activity towards pNPB, of induced bacterial cells with an OD<sub>600</sub>=0.1. Blue: Top10 induced with 0.5% arabinose, Orange: Top10 induced with 0.2% arabinose, Yellow: Top10 induced with 0.02% arabinose, Green: Top10 not induced ]]
 
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[[File:top_ten_induz(2).png|600px|center|thumb|graph shows the increasing velocity related to the level of induction]]
 
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==== Wednesday, 19.09.12 ====
 
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* activity assays on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar] worked well (incubation occured just for the night at 30°C)
 
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[[File:120919 top10 k808032 0.02 ara.jpg|200px]]
 
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[[File:120919 top10 k808032 0.2 ara.jpg|200px]]
 
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[[File:120919 top10 k808032 0.5 ara.jpg|200px]]
 
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* activity assays on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar]  with 0.5% mw L-arabinose for inducing
 
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** colonies 1-4 [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5 alpha], 5-8 [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], 9-12 [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]
 
-
** colonies 1,2,5,6,9,10 are carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 
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** colonies 3,4,7,8,11,12 are carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 
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==== Tuesday, 18.09.12 ====
 
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* activity assays on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar]  worked well
 
-
[[File:120920 k808032 k808030 dh5a top10 mg1655.tif.jpg|300px]]
 
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== Protein Expression ==
 
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=== CW 24 ===
 
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==== Thursday, 14.06.2012 ====
 
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* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] for protein expression of [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC]
 
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** each [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] is performed in 5 batches à 50 µL.
 
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** Parameter: T<sub>A</sub> = 57°C, t<sub>A</sub> = 35 s, t<sub>E</sub> = 65 s
 
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* [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
 
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#: 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]
 
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#: 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 ====
 
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*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] for quality control [[File:TU_Darmstadt_logo.png|200px]]
 
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=== CW 25 ===
 
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==== Wednesday, 20.06.2012 ====
 
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*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] for preparation [[File:TU_Darmstadt_logo.png|200px]]
 
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*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] products
 
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*: Concentration of produced [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence: 40 ng/µL
 
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==== Thursday, 21.06.2012 ====
 
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*[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.
 
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** Enzymes used: [https://2012.igem.org/Team:TU_Darmstadt/Materials/SfiI SfiI]
 
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** [https://2012.igem.org/Team:TU_Darmstadt/Materials/NEB_buffers#NEBuffer_4_.28green.29 NEBuffer: 4]
 
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** Digestion time: over night
 
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** Digestion temperature: 50°C
 
-
 
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==== Friday, 22.06.2012 ====
 
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*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX] vector
 
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*: Concentration range: 240-488 ng/µL
 
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*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX]
 
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** Enzymes used: [https://2012.igem.org/Team:TU_Darmstadt/Materials/SfiI SfiI]
 
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** NEBuffer: 4
 
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** Digestion time: 3 days
 
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** Digestion temperature: 50°C
 
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=== CW 26 ===
 
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==== Monday, 25.06.2012 =====
 
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*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] for quality control [[File:TU_Darmstadt_logo.png|200px]]
 
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*[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]]
 
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*: 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
 
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* [[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
 
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{| class="wikitable"
 
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|-
 
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! Component !! 1:3 !! 1:5
 
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|-
 
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| [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence || 1.12 µL || 2.2 µL
 
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|-
 
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| [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX pEX] || 0.64 µL || 0.64 µL
 
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|-
 
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| Ligase buffer || 4 µL || 4 µL
 
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|-
 
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| [https://2012.igem.org/Team:TU_Darmstadt/Materials/T4_DNA_Ligase T4 DNA ligase] || 1 µL || 1 µL
 
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|-
 
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| H<sub>2</sub>O || 33 µL || 30 µL
 
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|}
 
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* Ligation time: over night
 
-
 
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==== Tuesday, 26.06.2012 ====
 
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* [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.
 
-
 
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==== Wednesday, 27.06.2012 ====
 
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* 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 [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 ====
 
-
* [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
 
-
* [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 ===
 
-
==== 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 ====
 
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Purification_of_Periplasmatic_Proteins Purification of Periplasmatic Proteins] for all expression temperatures
 
-
 
-
==== 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"
 
-
|-
 
-
! 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
 
-
|-
 
-
| [[File:TU_Darmstadt_logo.png|200px]] || [[File:TU_Darmstadt_logo.png|200px]]
 
-
|}
 
-
* The best results were maintained at an expression temperature of 30°C
 
-
 
-
==== Friday, 06.07.2012 ====
 
-
* 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
 
-
 
-
=== CW 28 ===
 
-
==== Monday, 09.07.2012 ====
 
-
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep 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
 
-
* H<sub>2</sub>O: 7µL
 
-
 
-
==== Wednesday, 11.07.2012 ====
 
-
* [[Protein expression of FsC]] according to standard protocol
 
-
 
-
==== 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 ===
 
-
==== Monday, 16.07.2012 ====
 
-
* [[Protein expression of FsC]] according to standard protocol
 
-
 
-
==== 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 ===
 
-
==== Monday, 30.07.2012 ====
 
-
*[[Protein expression of FsC]] according to standard protocol
 
-
 
-
==== 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 ==  
Line 1,488: Line 1,179:
* 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]]
* 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
* now our lab can start with quantification of enzymatic activites of [[BBa_K808032]] on PET or model substrates
 +
 +
== Activity tests of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] ==
 +
=== Week 1 / CW 35 ===
 +
==== Friday, 31.08.12 ====
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5&alpha;] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] (arabinose inducable RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
 +
{| class="wikitable"
 +
|-
 +
! 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 ====
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5&alpha;] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 +
{| class="wikitable"
 +
|-
 +
! 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
 +
|}
 +
*  test tube 3: [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] without bacteria contains [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Kanamycin Kan], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Ampicillin AMP]
 +
* looks good but test tube 2 shows no significant change of colour
 +
==== Wednesday, 05.09.12 ====
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5&alpha;] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 +
{| class="wikitable"
 +
|-
 +
! 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
 +
|}
 +
* test tube 9: [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] without bacteria contains [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Kanamycin Kan], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Ampicillin AMP]
 +
* 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/GFP 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 [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 DH5&alpha;] 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%
 +
 +
==== Thursday, 06.09.12 ====
 +
*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay Activity assay] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5&alpha;] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 [BBa_K808032]] (arabinose inducable RBS-PhoA-His6tag-pNBEst13-Myctag-EstA)
 +
{| class="wikitable"
 +
|-
 +
! 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
 +
|}
 +
* test tube 11: [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT] without bacteria contains [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol CAM], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Kanamycin Kan], [https://2012.igem.org/Team:TU_Darmstadt/Materials/Ampicillin AMP]
 +
* all induced tubes turned yellow, even without PET-granula as a substrate
 +
 +
=== Week 3 / CW 37 ===
 +
==== Monday, 10.09.12 ====
 +
* we stopped the trial of quantification via PET-substrates because in terms of color the change of pH value (due to acid groups that are released by degradtion) is not very exactly. In order to get reproducible data, we are using new activity assays.
 +
* next bacterial assays are going to be in 96 well plates using a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#pNP-Assay pNP-Assay]
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Heat_Shock_Transformation Heatshock transformation] of [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay#LB-Agar-Tributyrin activity assay on LB-Tributyrin-CAM-plates] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/Arabinose L-arabinose] concentrations: 0.1%, 0.2%, 0.4%
 +
** colony 1-5 are [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5 alpha] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] but colony 6 is just DH5 alpha carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 +
** incubated over night at 37°C
 +
 +
==== Tuesday, 11.09.12 ====
 +
* inoculation of 5 mL [[DYT-media]]-CAM with K1 and K2 of transformed [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] from yesterday
 +
* acticity tests on [[LB-Tributyrin-CAM-plates]] shows good results
 +
** from now on incubation at room temperature
 +
[[File: 120911 dh5alpha k808032 0.1 ara.jpg|200px]]
 +
[[File: 120911 dh5alpha k808032 0.2 ara.jpg|200px]]
 +
[[File:120911 dh5alpha k808032 0.4 ara.jpg|200px]]
 +
==== Wednesday, 12.09.12 ====
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep#Promega_kit Plasmid preparation] of inoculated [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] colonies K1 and K2
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest DNA digestion] with EcoRI-HF & PstI-HF
 +
* qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis AGE]
 +
[[File:120912 Testrestriktion K808032 im ethylenglycol stamm K1 und K2 mit ecori und psti.tif]]
 +
==== Thursday, 13.09.12 ====
 +
* the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Activity_assay#LB-Agar-Tributyrin activity assay on LB-Tributyrin-CAM-plates] from tuesday gives good results
 +
 +
[[File:120913 dh5alpha k808032 0.1 ara.jpg|200px]] [[File:120913 dh5alpha k808032 0.2 ara.jpg|200px]] [[File:120913 dh5alpha k808032 0.4 ara.jpg|200px]]
 +
 +
* to perform a better expression of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030] we will use [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] as a host for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]. Its unability to metabolize L-arabinose is crucial for our decision. We hope to generate good expression levels.
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] (after washing ecletroporation cuvettes very carefully) of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 +
 +
==== Friday, 14.09.12 ====
 +
* yesterdays electroporation worked well
 +
* inoculation of 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT-media]-CAM with colony from plate of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 +
==== Saturday, 15.09.12 ====
 +
* making [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Glycerine_stock#Protocol 10% DMSO stocks] from 50 mL [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] culture
 +
 +
=== Week 4/ KW 38 ===
 +
==== Monday, 17.09.12 ====
 +
* inoculation of 4 x 50 mL [https://2012.igem.org/Team:TU_Darmstadt/Materials/DYT DYT-media]-CAM with [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] stocks
 +
* at OD<sub>600</sub>=0,5 the cultures are incduced with:
 +
** 0.02% mw L-arabinose
 +
** 0.2% mw L-arabinose
 +
** 0.5% mw L-arabinose
 +
** one culture is not induced and will serve as a negative control
 +
* after 3 hours an [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Antibody_staining antibody staining] is performed on our expressed [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] containing a myc-epitope
 +
* checking surface expression levels via [http://en.wikipedia.org/wiki/Flow_cytometry#Labels flow cytometry]
 +
** inducing worked well, we can go on with screening the enzymatic activity
 +
 +
[[Image:Flow cytometry 002 ara.png|200px]] [[Image:Flow cytometry 02 ara.png|200px]][[Image:Flow cytometry 05 ara.png|200px]] [[Image:Flow cytometry alle.png|200px]]
 +
 +
==== Tuesday, 18.09.12 ====
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030], serving as a negative  (directly used after 1h of incubation at 37°C in pure DYT-medium)
 +
* activity assay of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar] with L-arabinose concentrations: 0.02%, 0.2%, 0.5%
 +
** colonies 1-3 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] but colony 4 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Proteinpurification#Procedure Protein purification] from induced cultures and negative control
 +
** cell debris pellets are treated with [http://www.sigmaaldrich.com/catalog/product/sigma/d4641?lang=en&region=US n-Dodecyl ß-D-maltoside] as a detergent
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/SDS-Page#SDS-PAGE_.28Laemmli.29 SDS PAGE (Laemmli)] of pellets and supernatants
 +
[[Image: SDS Page of BBa_K808030.png|400px|thumb|left|2: supernatant 0.5% arabinose, 3: treated pellet 0.5% arabinose, 4: supernatant 0.2% arabinose, 5: treated pellet 0.2% arabinose, 7: supernatant 0.02% arabinose, 8: treated pellet 0.02% arabinose, 9: supernatant no arabinose, 10: treated pellet no arabinose ]]
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Western_Blot Westernblot] is successful as well
 +
* to quantify the hydrolytic activity of our induced bacteria we perform a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#Bacteria bacterial pNP-Assay]
 +
 +
[[File:top_ten_induz(geschwindigkeiten)(1).png|500px|center|thumb|graph shows absorbtion, ergo hydrolytic activity towards pNPB, of induced bacterial cells with an OD<sub>600</sub>=0.1. Blue: Top10 induced with 0.5% arabinose, Orange: Top10 induced with 0.2% arabinose, Yellow: Top10 induced with 0.02% arabinose, Green: Top10 not induced ]]
 +
[[File:top_ten_induz(2).png|600px|center|thumb|graph shows the increasing velocity related to the level of induction]]
 +
 +
==== Wednesday, 19.09.12 ====
 +
* activity assays on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar] worked well (incubation occured just for the night at 30°C)
 +
[[File:120919 top10 k808032 0.02 ara.jpg|200px]]
 +
[[File:120919 top10 k808032 0.2 ara.jpg|200px]]
 +
[[File:120919 top10 k808032 0.5 ara.jpg|200px]]
 +
* activity assays on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar]  with 0.5% mw L-arabinose for inducing
 +
** colonies 1-4 [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5 alpha], 5-8 [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], 9-12 [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]
 +
** colonies 1,2,5,6,9,10 are carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
 +
** colonies 3,4,7,8,11,12 are carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
 +
 +
==== Tuesday, 18.09.12 ====
 +
* activity assays on [https://2012.igem.org/Team:TU_Darmstadt/Materials/LB_Tributyrin-CAM-plates LB Tributyrin agar]  worked well
 +
[[File:120920 k808032 k808030 dh5a top10 mg1655.tif.jpg|300px]]
 +
 +
== Protein Expression ==
 +
=== CW 24 ===
 +
==== Thursday, 14.06.2012 ====
 +
* [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.
 +
** 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: [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: [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 ====
 +
*[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 ===
 +
==== 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 [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] sequence: 40 ng/µL
 +
 +
==== 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: [https://2012.igem.org/Team:TU_Darmstadt/Materials/SfiI SfiI]
 +
** [https://2012.igem.org/Team:TU_Darmstadt/Materials/NEB_buffers#NEBuffer_4_.28green.29 NEBuffer: 4]
 +
** Digestion time: over night
 +
** Digestion temperature: 50°C
 +
 +
==== 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
 +
*[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: [https://2012.igem.org/Team:TU_Darmstadt/Materials/SfiI SfiI]
 +
** NEBuffer: 4
 +
** Digestion time: 3 days
 +
** Digestion temperature: 50°C
 +
 +
=== CW 26 ===
 +
==== 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: [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 [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"
 +
|-
 +
! 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
 +
|-
 +
| [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
 +
|}
 +
* Ligation time: over night
 +
 +
==== 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 ====
 +
* 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 [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 ====
 +
* [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
 +
* [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 ===
 +
==== 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 ====
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Purification_of_Periplasmatic_Proteins Purification of Periplasmatic Proteins] for all expression temperatures
 +
 +
==== 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"
 +
|-
 +
! 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
 +
|-
 +
| [[File:TU_Darmstadt_logo.png|200px]] || [[File:TU_Darmstadt_logo.png|200px]]
 +
|}
 +
* The best results were maintained at an expression temperature of 30°C
 +
 +
==== Friday, 06.07.2012 ====
 +
* 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
 +
 +
=== CW 28 ===
 +
==== Monday, 09.07.2012 ====
 +
* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep 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
 +
* H<sub>2</sub>O: 7µL
 +
 +
==== Wednesday, 11.07.2012 ====
 +
* [[Protein expression of FsC]] according to standard protocol
 +
 +
==== 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 ===
 +
==== Monday, 16.07.2012 ====
 +
* [[Protein expression of FsC]] according to standard protocol
 +
 +
==== 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 ===
 +
==== Monday, 30.07.2012 ====
 +
*[[Protein expression of FsC]] according to standard protocol
 +
 +
==== 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]]
 +
 +
==Enzyme Activity Assays==
==Enzyme Activity Assays==
To characterise our BioBricks [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808026 BBa_K808026] (pNB-Est13) & [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808025 BBa_K808025] (FsC) a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#Enzyme pNP-assay]s are performed.
To characterise our BioBricks [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808026 BBa_K808026] (pNB-Est13) & [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808025 BBa_K808025] (FsC) a [https://2012.igem.org/Team:TU_Darmstadt/Protocols/pNP_Assay#Enzyme pNP-assay]s are performed.

Revision as of 06:28, 25 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.


SOE PCR

Week 1 / CW 17

Tuesday, 24.04.12

Wednesday, 25.04.12

Thursday, 26.04.12

Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE = 25 s

  1. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SKV
    primers: SKV a1 up XbaI & SKV a1 lo NdeI
  2. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
    primers: SOE A up & SOE a1 lo
  3. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with FsC
    primers: SOE A up & SOE a2 lo
  4. 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
  5. 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
  6. 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

120426 PCR 1-3 dunkel siehe Laborbuch 26.4.tif

  • 2-5 1.PCR, 6-9 2.PCR, 10-13 3.PCR

120426 PCR 4-6 siehe Laborbuch 26.4.tif

  • 2-5 4.PCR, 6-9 5.PCR, 710-13. 6.PCR

Friday, 27.04.12

Week 2 / CW 18

Monday, 30.04.12

120430 SOE PCR1 dunkel siehe Laborbuch 30.4.png

  1. 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
  2. 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

120430 PCR -2 der beiden EST Fragmente 1234 est lo 5678 est up.tif

Wednesday, 02.05.12

  1. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
    primers: SOE A up & SOE a1 lo
  2. 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
  3. 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

120502 PCR 1.png 120502 PCR 2 und 3.png

TA1 = 60°C, ta1 = 25 s, tE1 = 25 s
TA = 60°C, ta2 = 25 s, tE2 = 35 s

Tuesday, 03.05.12

Friday, 04.05.12

  1. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
    primers: SOE A up & SOE a1 lo
  2. 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
  3. 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
  4. PCR on pEST100
    gene of interest: EstA part2 for SOE PCR with EstA part1
    primers: SOE EstA mut PstI out up & SOE D lo

120504 pcr Esta1 Est13 + EstA1 Fsc.png 120504 pcr Esta2.png 120504 pcr PhoAEst13lapp.png

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

  1. 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
  2. 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
  3. 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
  4. 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
  5. 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

Week 4 / CW 20

Monday, 14.05.12

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

Tuesday, 15.05.12

  1. 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
  2. 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

Week 5 / Kw 21

Monday, 23.05.12

  1. 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
  2. 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

Wednesday, 23.05.12

  1. 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

120524 SOE PCR EstAFsc.png

Thursday, 24.05.12

  1. PCR on EstA for SOE PCR with pNB-Est13 from yesterday
    gene of interest: EstA for SOE PCR with pNB-Est13
    primers: SOE c1 up & SOE D lo
  2. PCR on EstA for SOE PCR with FsC
    gene of interest: EstA for SOE PCR with FsC
    primers: SOE c2 up & SOE D lo
  3. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
    primers: SOE A up & SOE a1 lo
  4. 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

Week 6 / Kw 22

Tuesday, 29.05.12

  1. 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
  2. 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
  3. 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

Thursday, 29.05.12



Due to better results in SKV we stopped our tries in SOE PCR assembly of our total construct BBa_K808032

SKV

Week 1 / CW 17

Tuesday, 24.04.12

Wednesday, 25.04.12

Thursday, 26.04.12

Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE = 25 s

  1. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SKV
    primers: SKV a1 up XbaI & SKV a1 lo NdeI

120426 PCR 1-3 siehe Laborbuch 26.4.tif

Friday, 27.04.12

Week 2 / CW 18

Monday, 30.04.12

120430 Testrestrikt.XbaINdeI.tif

TU Darmstadt logo.png

Week 4/CW 20

Monday, 07.05.

Annotation: If it does not say anything else, Ligation is always done in 20µl batches.

Tuesday, 08.05.

Wednesday, 09.05.

Thursday, 10.05.

Friday, 11.05.

  • an analytic, 1% agarose-gel proves that the amplification of FSC, Est13 and EstA was successfull.

TU Darmstadt logo.png

  • 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.

Tuesday, 15.05.

Wednesday, 16.05.

Friday, 18.05.

Week 6/CW 22

Monday, 21.05.

Tuesday, 22.05.

Wednesday, 23.05.

Wednesday, 23.05.

Thursday, 24.05.

Friday, 25.05.

week 7/CW 23

Tuesday, 29.05.

Wednesday, 30.05.

Thursday, 31.05.

Friday, 01.06.

week 8/CW 24

Monday, 04.06.

Tuesday, 05.06.

Wednesday, 06.06.

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.

Tuesday, 12.06.

Wednesday, 13.06.

Friday, 15.06.

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.

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
  1. PCR on pEST100
    gene of interest: PhoA for designing part BBa_K808028
    primers: BBa PhoA up & BBa PhoA down
  2. 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
  3. 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
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

120718 SOE PCR Est13.jpg

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

120718 EstA mit bba.jpg

Thursday, 19.07.12

    1. PhoA from Tuesday, 17.07.12
    2. pNB-Est13 fromTuesday, 17.07.12
    3. EstA from Wednesday, 18.07.12
    4. pSB1C31
    5. pSB1C32

Friday, 20.07.12

Week 2 / CW 30

Monday, 23.07.12

120723 BBaPhoA BBa PhoA BBa FsC BBa FsC BBa Est13 BBa Est13 BBa Est13.jpg

Tuesday, 24.07.12

Wednesday, 25.07.12

120725 Colony BBaEstA positiv probe auf SKV EstA.jpg

Thursday, 26.07.12

Friday, 27.07.12

Week 3 / CW 31

Monday, 30.07.12

Tuesday, 31.07.12

Trouble shooting

  • 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


Wednesday, 01.08.12

  • due to change of strategy the following PCR 1s are performed
  1. PCR on pEST100
    gene of interest: PhoA BioBrick for assembly of part BBa_K808028
    primers: BBa PhoA up & BBa PhoA down
  2. 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
  3. PCR on pEST100
    gene of interest: EstA part2 for SOE PCR of EstA (BBa_K808027)
    primers: BBa EstA mut up & BBa EstA down
  4. 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
  5. 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

120801 soe pcr esta1 pcr phoa.jpg

120801 soe pcr est131 est132.jpg

120801 soe pcr esta2.jpg

  1. SOE PCR
    gene of interest: EstA
    primers: EstA part1 & EstA part2
  2. 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

    1. PhoA
    2. pNB-Est13
    3. EstA
    • enzymes used: EcorI-HF & PstI-HF
    • in NEBuffer 4
    • each digestion is performed in a 60 µL batch
    • Digestion time: 1.5 h

Thursday, 02.08.12

Friday, 03.08.12

  • transformation succeeded
    • colonies picked to inoculate 5 ml DYT-media-CM

Saturday, 04.08.12

TA = 60°C, ta = 35s, tE = 25 s

120804 Verdau PhoA, EstA, Est13, PCR auf EstA, Est 13, PhoA, Kontrolle auf Insert und Vektor.jpg

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:

Monday, 06.08.12

Tuesday, 07.08.12

Wednesday, 08.08.12

120808 Restriktion mit E BsaI PhoA Est13 und bsai s EstA.jpg

Thursday, 09.08.12

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
  • Colony PCR on picked colonies
    • Annotation: TA = 55°C, tA = 25 s, tE = 2 min, done with house-taq so its similiar to PCR 2
    • primers: VR & VF2
  • Ligation and transformation succeeded

120811 PCR (VR,VF2) und Verdau (EcoRI,PstI) auf BBa Gesamt von Geneart.jpg

Week 5 / CW 33

Monday, 13.08.12

  1. 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
  2. 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
  3. 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

120813 PCR 1-4 RBSPE 5-8 EXN-Myc-Est 9-12.jpg

120813 PCR Q5 auf bba gesamt konstrukt.jpg

  1. 1.PCR & 2.PCR in pSB1C3 carrying RFP
  2. 3.PCR in pSB1C3 carrying RFP
  3. 1.PCR & 2. PCR in BBa_K808000 possibility 1
  4. 3.PCR in BBa_K808000 possibility 1
  5. 1.PCR & 2. PCR in BBa_K808000 possibility 2
  6. 3.PCR in BBa_K808000 possibility 2

Tuesday, 14.08.12

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

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:

Conclusion:

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

Week 6 / CW 34

Monday 20.08.12

Tuesday 21.08.12

TA = 55°C, tA = 25 s, tE = 5 min
primers: VF2 & VR

Wednesday, 22.08.12

Verdau vom Gesamtkonstrukt 4.2_2.2 und 5.1_1.1 beide temperaturen.tif

Thursday, 23.09.12

Friday, 24.08.12

Week 7 / CW 35

Monday, 27.08.12

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

TU Darmstadt logo.png

  • 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

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
  • test tube 3: DYT without bacteria contains CAM, Kan, AMP
  • 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
  • test tube 9: DYT without bacteria contains CAM, Kan, AMP
  • 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%

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
  • test tube 11: DYT without bacteria contains CAM, Kan, AMP
  • all induced tubes turned yellow, even without PET-granula as a substrate

Week 3 / CW 37

Monday, 10.09.12

  • we stopped the trial of quantification via PET-substrates because in terms of color the change of pH value (due to acid groups that are released by degradtion) is not very exactly. In order to get reproducible data, we are using new activity assays.
  • next bacterial assays are going to be in 96 well plates using a pNP-Assay
  • Heatshock transformation of [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
  • activity assay on LB-Tributyrin-CAM-plates with L-arabinose concentrations: 0.1%, 0.2%, 0.4%
    • colony 1-5 are [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5 alpha] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] but colony 6 is just DH5 alpha carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
    • incubated over night at 37°C

Tuesday, 11.09.12

  • inoculation of 5 mL DYT-media-CAM with K1 and K2 of transformed [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655] from yesterday
  • acticity tests on LB-Tributyrin-CAM-plates shows good results
    • from now on incubation at room temperature

120911 dh5alpha k808032 0.1 ara.jpg 120911 dh5alpha k808032 0.2 ara.jpg 120911 dh5alpha k808032 0.4 ara.jpg

Wednesday, 12.09.12

120912 Testrestriktion K808032 im ethylenglycol stamm K1 und K2 mit ecori und psti.tif

Thursday, 13.09.12

120913 dh5alpha k808032 0.1 ara.jpg 120913 dh5alpha k808032 0.2 ara.jpg 120913 dh5alpha k808032 0.4 ara.jpg

  • to perform a better expression of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030] we will use [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] as a host for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]. Its unability to metabolize L-arabinose is crucial for our decision. We hope to generate good expression levels.
  • Electroporation (after washing ecletroporation cuvettes very carefully) of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]

Friday, 14.09.12

  • yesterdays electroporation worked well
  • inoculation of 50 mL DYT-media-CAM with colony from plate of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]

Saturday, 15.09.12

  • making 10% DMSO stocks from 50 mL [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] culture

Week 4/ KW 38

Monday, 17.09.12

  • inoculation of 4 x 50 mL DYT-media-CAM with [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] stocks
  • at OD600=0,5 the cultures are incduced with:
    • 0.02% mw L-arabinose
    • 0.2% mw L-arabinose
    • 0.5% mw L-arabinose
    • one culture is not induced and will serve as a negative control
  • after 3 hours an antibody staining is performed on our expressed [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] containing a myc-epitope
  • checking surface expression levels via [http://en.wikipedia.org/wiki/Flow_cytometry#Labels flow cytometry]
    • inducing worked well, we can go on with screening the enzymatic activity

Flow cytometry 002 ara.png Flow cytometry 02 ara.pngFlow cytometry 05 ara.png Flow cytometry alle.png

Tuesday, 18.09.12

  • Electroporation of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030], serving as a negative (directly used after 1h of incubation at 37°C in pure DYT-medium)
  • activity assay of [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10] containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] on LB Tributyrin agar with L-arabinose concentrations: 0.02%, 0.2%, 0.5%
    • colonies 1-3 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] but colony 4 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]
  • Protein purification from induced cultures and negative control
    • cell debris pellets are treated with [http://www.sigmaaldrich.com/catalog/product/sigma/d4641?lang=en&region=US n-Dodecyl ß-D-maltoside] as a detergent
  • SDS PAGE (Laemmli) of pellets and supernatants
2: supernatant 0.5% arabinose, 3: treated pellet 0.5% arabinose, 4: supernatant 0.2% arabinose, 5: treated pellet 0.2% arabinose, 7: supernatant 0.02% arabinose, 8: treated pellet 0.02% arabinose, 9: supernatant no arabinose, 10: treated pellet no arabinose
















graph shows absorbtion, ergo hydrolytic activity towards pNPB, of induced bacterial cells with an OD600=0.1. Blue: Top10 induced with 0.5% arabinose, Orange: Top10 induced with 0.2% arabinose, Yellow: Top10 induced with 0.02% arabinose, Green: Top10 not induced
graph shows the increasing velocity related to the level of induction

Wednesday, 19.09.12

  • activity assays on LB Tributyrin agar worked well (incubation occured just for the night at 30°C)

120919 top10 k808032 0.02 ara.jpg 120919 top10 k808032 0.2 ara.jpg 120919 top10 k808032 0.5 ara.jpg

  • activity assays on LB Tributyrin agar with 0.5% mw L-arabinose for inducing
    • colonies 1-4 [http://ecoliwiki.net/colipedia/index.php/DH5_alpha DH5 alpha], 5-8 [http://ecoliwiki.net/colipedia/index.php/TOP10 Top10], 9-12 [http://ecoliwiki.net/colipedia/index.php/MG1655 Mg1655]
    • colonies 1,2,5,6,9,10 are carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]
    • colonies 3,4,7,8,11,12 are carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808030 BBa_K808030]

Tuesday, 18.09.12

120920 k808032 k808030 dh5a top10 mg1655.tif.jpg

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
  1. 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

CW 25

Wednesday, 20.06.2012

Thursday, 21.06.2012

Friday, 22.06.2012

CW 26

Monday, 25.06.2012 =

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

Wednesday, 27.06.2012

  • Two positive clones on LB CAM plates picked for liquid culture
    50 mL DYT CAM cultures at 180 rmp and 37°C, over night

Thursday, 28.06.2012

CW 27

Monday, 02.07.2012

Tuesday, 03.07.2012

Wednesday, 04.07.2012

Expression at 16°C Expression at 25°C
TU Darmstadt logo.png TU Darmstadt logo.png
Expression at 30°C Expression at 37°C
TU Darmstadt logo.png TU Darmstadt logo.png
  • The best results were maintained at an expression temperature of 30°C

Friday, 06.07.2012

CW 28

Monday, 09.07.2012

Wednesday, 11.07.2012

Thursday, 12.07.2012

CW 29

Monday, 16.07.2012

Tuesday, 17.07.2012

CW 31

Monday, 30.07.2012

Tuesday, 31.07.2012


Enzyme Activity Assays

To characterise our BioBricks [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808026 BBa_K808026] (pNB-Est13) & [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808025 BBa_K808025] (FsC) a pNP-assays are performed.

CW 37

Est13

  • a pNP-assayis performed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808026 BBa_K808026] (pNB-Est13)
Lineweaver-Burk plot of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808026 BBa_K808026]
Km and Vmax calculation

To find Km the growth of the first seven data points were written down in a diagram. The slope of the regression line gave the speed of the hydrolysis for the single substrate concentrations. Now the value of the speed with the associated concentration of substrate were plotted in a Lineweaver-Burk diagram. At the point where the regression line is crossing the x-axis the x-coordinate amounts to -1/Km. So Km is about 400µM. To calculate the value of Vmax we used the point where the plotted line crosses the y-axis. The associated y-coordinate is 1/Vmax. Consequently Vmax for 50nM Est13 is 0,002798 1/s.

Kcat calculation

The value Kcat is calculated through dividing Vmax by the enzyme concentration of 50nM. Kcat counts 0,055951 1/mol*s.

FsC

  • a pNP-assayis performed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808026 BBa_K808025] (FsC)
Lineweaver-Burk plot of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808025 BBa_K808025
Km and Vmax calculation

To find Km the growth of the first twelve data points were written down in another diagram. The slope of the regression line gave the speed of the hydrolysis for the single substrate concentrations. Now the value of the speed with the associated concentration of substrate were plotted in a Lineweaver-Burk diagram. At the point where the regression line is crossing the x-axis the x-coordinate amounts to -1/Km. So Km is about 400µM. To calculate the value of Vmax we used the point where the plotted line crosses the y-axis. The associated y-coordinate is 1/Vmax. Consequently Vmax for 5nM FsC is 0,001467 1/s.

Kcat calculation

The Kcat value is calculated through dividing Vmax by the enzyme concentration of 5nM. Kcat counts 0,293395 1/mol*s.

FsC with ehtylenglycol

Our Simulation lab was able to show , theoratically, a relation between an decrease of enzyme activity and an increase of ethylenglycol concentration. This could be a hint for the mysterious loss of hydrolysis when the degradation rate reaches 5% of PET foil weight as it is described in the literature (Ronkvist, Å. M., Xie, W., Lu, W., & Gross, R. a. (2009). Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate). Macromolecules, 42(14), 5128–5138. doi:10.1021/ma9005318). We tried to proof their theory of potentially higher ethylenglycol concentration surrounding the PET foil. An increasing amount of ehtylenglycol should hamper the hydrolytic activity of FsC in a pNP-assay.

Procedure

The lab test procedure was nearly equal to the normal kinetics records with only one difference. The reaction solution consisted of different composites of PBS buffer and ethylenglycol (0 - 20%). All records were done at a concentration of 1mM of 4-Nitrophenyl butyrate in the experiment.

Records of the different ethylenglycol concentrations and the associated speeds of hydrolisis in a diagram
of enzyme activity related to an increasing ehtylenglcol concentration
Interpretation

As you can see there is a stagnation of the speed of hydrolysis of the ester caused by an increasing concentration of ethylenglycol. But by that you cannot tell that ethylenglycol inhibits the FsC, because the hydrolysis of the ester rose with a higher concentration of ethylenglycol in the negative samples. So it could be that the ethylenglycol starts a concurrent reaction or blocks the 4-Nitrophenyl butyrate.

Plot of "activty" in the negative sample: pNPB & ethylenglycol