Team:TU Darmstadt/Labjournal/Degradation

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We stopped using SOE PCR after a couple of weeks. It is a good method to perform mutationial PCR but to assemble genes of the length of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] it is not the best. Due to bad yields of gel extraction after the first assembly steps we stopped SOE PCR and went on with [[SKV]]. But eventually we got our chimeric genes synthesized by [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/gene-synthesis/GeneArt-Gene-Synthesis.html GeneArt].

** [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEST100 pEST100], carrying a CMP-resistance, the genes of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808028 phoA], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808025 FsC] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808027 EstA]]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of  the 3 overnigth [[http://ecoliwiki.net/colipedia/index.php/Category:Strain:BL21 Bl21] cultures and [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Quantification_/_NanoDrop concentration meassurement via Nanodrop]

#: gene of interest: Promo-LacO-RBS-Phoa for [[https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#SKV SKV]

* [[https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR SOE PCR]  

* [[PCR 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]]

# PCR on [[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC]

#: gene of interest: [[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC]

#: primers: [[SOE c2 up]] & [[SOE D lo]]

# PCR on [[pEST100]]

#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]]

#: primers: [[SOE A up]] & [[SOE a1 lo]]

# PCR on [[pEST100]]

#: gene of interest: [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[EstA part1]]

#: primers: [[SOE b2 up]] & [[SOE b2 lo]]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction]

** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=39 ng/µL

** c([[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])=20 ng/µL

** c([[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]])=13 ng/µL

* [[SOE PCR]]s

# [[SOE PCR]]

#: gene of interest: [[Promo-LacO-RBS-Phoa-Fsc-EstA]]

#: primers: [[SOE A up]] & [[SOE D lo]]

#: template: [[EstA]] from [[Thursday, 24.05.12]] & [[Promo-LacO-RBS-Phoa-Fsc]] from [[Wednesday, 16.05.12]]

# [[SOE PCR]]

#: gene of interest: [[Promo-LacO-RBS-Phoa-pNBEst13-EstA]]

#: primers: [[SOE A up]] & [[SOE D lo]]

#: template: [[EstA]] from [[Thursday, 24.05.12]] & [[Promo-LacO-RBS-Phoa]] from [[Wednesday, 02.05.12]] & [[pNB-Est13]] from [[Monday, 14.05.12]]

# [[SOE PCR]]

#: gene of interest: [[Promo-LacO-RBS-Phoa-pNBEst13]] for [[SOE PCR]] with [[EstA]]

#: primers: [[SOE A up]] & [[SOE b1 lo]]

#: template: [[Promo-LacO-RBS-Phoa]] from [[Wednesday, 02.05.12]] & [[pNB-Est13]] from [[Monday, 14.05.12]]

** [[Promo-LacO-RBS-Phoa-Fsc-EstA]] worked

** [[Promo-LacO-RBS-Phoa-pNBEst13]] worked

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction]

** c([[Promo-LacO-RBS-Phoa-Fsc-EstA]])= 8 ng/µL

** c([[Promo-LacO-RBS-Phoa-pNBEst13]])= 13 ng/µL

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

** [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEST100 pEST100], carrying a CM-resistance, the genes of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808028 phoA], [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808027 EstA]

# PCR on pEST100

* DNA digestion of [[pET16b]]

* Amplification of the genes FSC, Est13 and EstA via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR]

** 1-4= Est13

** 5-8= EstA

** 9-10= FSC  

*[[File: 120515_colony_PCR_+_SkV_Est13_beschriftet.png]]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] with NdeI and NcoI of the plasmids ([[PhoA]] x [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b]) 1-4, so FSC and Est13 can be added to the construct.

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] of EST13  

* [[File: 120516_SOE_PCR_PhoA_Est13_+_SkV_Est13_FsC2.png]]

** 1-4 = Est 13

*(phoA x pET16b(+))= 3,72 ng/µl

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Colony_PCR Colony-PCR] proves, that Est13 has been inserted into ([[phoA]] x [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b])

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] is performed again to amplify FSC and Est13

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [[FSC]] and [[Est13]] is cleaned up via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]

* [[MIniprep]] of ([[Est13]] x ([[phoA]] x [[pET16b]]) via Promega-Kit

* [[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b]])

* [[Electroporation]] of DH5alpha with 5µl of the [[LIgaton]]: [[FSC]] x ([[phoA]] x [[pET16b]])

* [[PCR 1]] to amplify [[EstA]], [[(Thursday, 10.05.)]]

** An analytical [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] is performed

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [[EstA]] (product of PCR, 80 µl) and Plasmid: ([[Est13]] x [[phoA]] x [[pET16b]])  

** The [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] shows, that two DNA fragments are cut out of the vector ([[Est13]] x [[phoA]] x [[pET16b]]). This might be a hint that [[Est13]] is not inserted.

** 1-3 = ([[Est13]] x [[phoA]] x [[pET16b]])

** 4-5 = [[EstA]]

** c([[EstA]]) = 6,7 ng/µl

** c([[Est13]] x [[phoA]] x [[pET16b]])= 23 ng/µl  

* [[Electroporation]] of ([[FSC]] x [[phoA]] x [[pET16b]]) worked well, plates are covered with colonies which are picked to be grown in 5ml DYT/Amp medium over night.

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the amplified ([[FSC]] x [[phoA]] x [[pET16b]])

** [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] to ligate [[EstA]]

* to be sure that the plasmid ([[Est13]] x [[phoA]] x [[pET16b]]) really carries [[Est13]] and [[PhoA]], an analytic [[PCR1]] is performed. Also [[pEt16b]] is used as template with different primers.

** [[pEt16b]] [[Wednesday, 25.04.]]

** ([[phoA]] x [[pET16b]]) [[Wednesday, 16.05.]]

** ([[Est13]] x [[phoA]] x [[pET16b]]) [[Monday,29.05.]]

* primers, used on each template:

** [[SKV b1 up NdeI]], [[SKV b1 lo NcoI]]

** [[SKV a1 up XbaI]], [[SKV a1 lo NdeI]]

* to solve the problem, the [[LIgation]] of [[FSC]] and [[Est13]] shall be repeated from the beginning.

* Additionally,the decision was to go on with the Ligation of [[EstA]] into the Plasmids ([[Est13]] x [[phoA]] x [[pET16b]]) and ([[FSC]] x [[phoA]] x [[pET16b]]), in case that the PCR was proceeded under wrong conditions.

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of ([[phoA]] x [[pET16b(+)]])

* [[LIgation]] of [[Est13]] and [[FSC]]x([[PhoA]]x[[pET16b(+)]])

** c([[Est13]])= 3 ng/µl

** c([[FSC]])= 11 ng/µl

** c([[[[phoA]] x [[pET16b(+)]])= 29 ng/µl

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of ([[FSC]]x ([[PhoA]] x [[pET16b(+)]])

=== week 8/CW 24 ===

* restriction of  ([[FSC]]x([[PhoA]]x[[pET16b(+)]]) and ([[phoA]]x[[pET16b(+)]]) is cleaned up by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]

**c([[FSC]]x ([[PhoA]] x [[pET16b(+)]]) = 14 ng/µl

*[[Electroporation]] of [[DH5alpha]] with [[Ligation]]: ([[FSC]]x[[PhoA]]x[[pET16b(+)]]), ([[EST13]]x[[PhoA]]x[[pET16b(+)]]) [[Friday, 01.06]]

*[[colony-PCR]] on [[EST13]] & [[FSC]] is negative.

* [[Electroporation]] of [[DH5alpha]] with [[Ligation]]-assays from 01.06.

* [[Ligation]] of [[EstA]][[Wednesday,30.05]] and the Plasmids ([[Est13]]x[[phoA]]x[[pET16b(+)]]),([[FSC]]x[[phoA]]x[[pET16b(+)]]) from [[Monday,04.06.]]

**[[Electroporation]] of [[DH5alpha]] with ([[EstA]]x[[Est13]]x[[phoA]]x[[pET16b(+)]]) and ([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]])

=== week8/ CW24 ===

* all plates are covered with cells, so a [[colony-PCR]] is performed on either [[Est13]],[[FSC]] or [[EstA]]

* primer:

** [[SKV b1 up NdeI]], [[SKV b1 lo NcoI]]

** [[SKV b2 up NdeI]], [[SKV b2 lo NCOI]]

** [[SKV c1 up NcoI]], [[SKV c1 lo EcoRI]]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] shows, that EstA has been inserted in both Plasmids, ([[Est13]]x[[PhoA]]x[[pET16b(+)]]) and ([[FSC]]x[[PhoA]]x[[pET16b(+)]])

* colonies No.3,4 ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]) and No.15,16 ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]) seem to be positive on EstA and are picked to be grown in 5ml DYT/Amp medium.

*[[colony-PCR]] on [[Est13]] and [[FSC]] shows a lot of unspecific bands, so cloning of the genes from the beginning has to be dropped.

*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the clones No. 3,4,15 and 16. [[Tuesday, 12.06.]]

** c([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]])=

** c([[EstA]]x[[Est13]]x[[phoA]]x[[pET16b(+)]])=

* for gene-expression, it is performed a [[Elektroporation]] of [[BL21]]-cells with 1µl of the PLasmid-DNA which has just been isolated.

* Transformation of [[BL21]] worked well

* [[Tributyrinagar plates]] with 1mM IPTG were generated, to induce the Lac-Promotor of [[pET16b(+)]], and to demonstrate the esterase-activity by the lysis of Tributyrin.

* afterwards [[Tributyrinagar plates]] are inoculated with cells which were transformed by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] No.3,4,15,16 on [[Wednesday, 13.06.]]

=== week9/CW25 ===

* [[Tributyrinagar plates]] are covered with cells, but no lysis is visible.

* The plasmids No.3,4 ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]) and No.15,16 ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]) are prepared for sequencing.

* primer:

* ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]):

** [[T7 up]]

** [[T7 lo]]

** [[SOE EstA mut up lo]]

** [[SOE EstA mut up lo]]

* ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]):

** [[T7 up]]

** [[T7 lo]]

** [[SOE Est13 mut up lo]]

** [[SOE Est13 mut up lo]]

It was discovered later, that there was a contamination of the [[DH5alpha]] cells with other plasmid-DNA, probably caused by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation].  

[[Verlinkung Arne]]

The contamination of the cells may have also caused false-positive results of the [[colony-PCR]]s.  

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.

=== Week 1 / Kw 29 ===

* [[PCR 1]]

# PCR on [[pEST100]]  

#: gene of interest: [[PhoA]] for designing part [[BBa_K808028]]  

# PCR on [[pET26b(+)]]

#: gene of interest: [[pNB-Est13 part1]] for designing part [[BBa_K808026]] via [[SOE PCR]]

# PCR on [[pET26b(+)]]

* preparative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis]

* [[SOE PCR]]

* qualitative [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of [[SOE PCR]] (from [[Tuesday, 17.07.12]])

* [[PCR 1]]  

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR] product

* inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])

## [[PhoA]] from [[Tuesday, 17.07.12]]

## [[pNB-Est13]] from[[Tuesday, 17.07.12]]

## [[EstA]] from [[Wednesday, 18.07.12]]

## [[pSB1C3]]1

## [[pSB1C3]]2

** enzymes used: [[EcorI-HF]] & [[PstI-HF]] / for  [[pSB1C3]]2 [[SpeI]] is used instead of [[PstI]]

** in NEBuffer 4

* [[Ligation]] into digested and 5' dephosphorylated [[pSB1C3]] 1

** [[pNB-Est13]]

** [[PhoA]]

** [[EstA]]

* [[bacterial transformation]] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [[DH5alpha]]

**[[Ligation]] into digested and 5' dephosphorylated [[pSB1C3]]

*** [[pNB-Est13]]

*** [[PhoA]]

* inoculation of 5 mL [[DYT-media]] with 5 µL CAM and two positive colonies from each transformation (from pNB-Est13 3 colonies are taken) .

** in addition [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] is already on plate, but to controle it, 2 [[colony PCR]]s are performed

* [[Ligation]] of [[EstA]] into digested and 5' dephosphorylated [[pSB1C3]]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [[DH5alpha]]

* inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])

=== Week 2 / CW 30 ===

* [[Colony PCR]] of picked colonies from [[Friday, 20.07.12]]

* 2-3 [[PhoA]] colonies, 4-5 [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] colonies, 6-8 [[pNB-Est13]] colonies

** c([[PhoA]] in [[pSB1C3]])=220 ng/µL

** c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL

* inoculation of 2 x 5 mL [[DYT-media]]-CAM with [[pSB1C3]] carrying [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC]

* second transformation of [[EstA]] from [[Friday, 20.07.12]] worked, but too many colonies!  

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of the 2 [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] [[DYT-media]]-CAM cultures with [[pSB1C3]] carrying [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] from yesterday

** c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=168 ng/µL

* purity plate of [[EstA]] in [[pSB1c3]] is positive

** [[Colony PCR]] with positive control by amplfifying an [[SKV]]-[[EstA]]

** Annotation: T_A = 57°C, t_A = 25 s, t_E = 2 min, done with NEB- Phusion so its similiar to [[PCR 1]]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] of [[Colony PCR]]

* 2 [[EstA]] colony, 4 [[EstA]] from SKV as a control

* [[ Miniprep]] of inoculated 5 ml [[DYT-media]]-CAM

** c([[EstA]] in [[pSB1C3]])=350 ng/µL

** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VR]], 9 µL ddH₂O

** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VF2]], 9 µL ddH₂O

** 7 µL of c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=168 ng/µL, 1µL [[VR]], 7 µL ddH₂O

** 7 µL of c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=168 ng/µL, 1µL [[VF2]], 7 µL ddH₂O

** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VR]], 9 µL ddH₂O

** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VF2]], 9 µL ddH₂O

** 5 µL of c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL, 1µL [[VR]], 9 µL ddH₂O

** 5 µL of c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL, 1µL [[VF2]], 9 µL ddH₂O

* in order to prepare [[DMSO stocks]] and [[Minipreps]] 5 mL [[DYT-media]]-CAM are inoculated with following colonies containing

** [[pNB-Est13]] in [[pSB1C3]]

** [[PhoA]] in [[pSB1C3]]

** [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]]

** [[EstA]] in [[pSB1C3]]

* inoculated [[pNB-Est13]] in [[pSB1C3]] from [[Thursday, 26.07.12]] has not grown over night

** new colony pcked from plate and incubated in 5 mL [[DYT-media]]-CAM at 37°C over night

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of 3 mL from the following over night [[DYT-media]] cultures from [[Thursday, 26.07.12]]

** c([[PhoA]] in [[pSB1C3]])=440 ng/µL

** c([[pNB-Est13]] in [[pSB1C3]])=450 ng/µL

** c([https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]])=90 ng/µL

=== Week 3 / CW 31 ===

* [[bacterial transformation]] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation] of [[DH5alpha]] with c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL from [[Monday, 23.07.12]]

** very much transformants on crossed out plates after [[bacterial transformation]] by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation]

** at first [[Colony PCR]] is positive

** second [[Colony PCR]] is negative

** BUT [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] in [[pSB1C3]]  

** our [[bacterial transformation]] is done by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation]

** this slow decrease of plasmid could explain the missing second positive signal of [[Colony PCR]]

** switching our method from [[Electroporation]] to [[Heatshock transformation]]

 

* due to change of strategy the following [[PCR 1]]s are performed

# PCR on [[pEST100]]

# PCR on [[pEST100]]

# PCR on [[pEST100]]

# PCR on [[pET26b(+)]]

# PCR on [[pET26b(+)]]

* [[SOE PCR]]s

** enzymes used: [[EcorI-HF]] & [[PstI-HF]]  

** in NEBuffer 4

* [[Heatschock transformation]] of [[DH5alpha]] with our ligation batches

* transformation succeeded

** colonies picked to inoculate 5 ml [[DYT-media]]-CM

* test [[DNA Digestion]] with EcoRI-HF and PstI-HF

* tests succeeded

* retransformation of [[DH5alpha]] by [[Heatshock transformation]] with the following plasmids with 1 µL each

=== Week 4 / Kw 32 ===

* [[Heatshock transformation]] of [[DH5alpha]] with 1 µL synthesis products (c=100 ng/µL):

* [[Colony PCR]] on 4 colonies of each transformation of synthesis product from [[Monday, 06.08.12]]

* [[Colony PCR]] succeeded

* inoculation of 5 ml [[DYT-media]]-KAN with colonies

* [[Heatshock transformation]]  of [[DH5alpha]] with 10 µL of each ligation batch from yesterday

** testing ligation by [[DNA Digestion]] of plasmids with [[EcorI-HF]] & [[PstI-HF]]

* [[Colony PCR]] on picked colonies

** primers: [[VR]] & [[VF2]]

* Ligation and transformation succeeded

* 2-5 [[bba prefix-PhoA-His6tag-pNBEst13-Myctag-EstA- bba suffix]] running at round about 3 kb  

=== Week 5 / CW 33 ===

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/PCR PCR]s succeeded

* [[PCR clean up]] of 1.-3. PCR

* [[DNA Digestion]] of [[pSB1C3]] carrying [[RFP]] part [[????]]

** enzymes used: [[XbaI]] & [[PstI]]

* [[DNA Digestion]] of part [[BBa_K808000]] ([[pSB1C3]] carrying [[Arabinose inducible promotor]])

** enzymes used: [[SpeI]] & [[PstI]]

* [[Ligation]] at 4°C over night of the following combinations:

* [[Heatshock transformation]] of ligation batches 1-6 from yesterday

* transformation succeeded

* inoculation of 5 mL [[DYT-media]]-CAM of the following colonies

* [[Colony PCR]] on colonies 1.1-6.2 with house-taq  

** primers: [[VR]] & [[VF2]]

* [[DNA Digestion]] of preped colonies with [[EcorI-HF]] & [[PstI-HF]]

* new inoculation of 5 mL [[DYT-media]]-CAM of Colonies 1.1 - 5.2

* colony 1.1: 1.PCR & 2.PCR in [[pSB1C3]]

** should be RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808030]]) in [[pSB1C3]] (length: ~ 3.1 kb)

* colony 2.1: 3.PCR in [[pSB1C3]] upstream of part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1

** should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808032]]) in [[pSB1C3]] (length: ~ 4.3kb)

* colony 3.1: 3.PCR in [[pSB1C3]] upstream of part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 2

** should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808032]]) in [[pSB1C3]] (length: ~ 4.3kb) like colony 2.1

* colony 4.1: 3.PCR in [[pSB1C3]]

* colony 5.1: 1.PCR & 2.PCR in [[pSB1C3]] upstream of part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1

** should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808032]]) in [[pSB1C3]] (length: ~ 4.3kb)

* digestion with [[EcoRI-HF]] & [[PstI-HF]] of all [[ligations]] from [[Thursday 16.08.12]] in part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1 shows bands at round about 3 - 3.5 kb

=== Week 6 / CW 34 ===

* [[Ligation]] of [[BBa_K808000]] upstream of [[BBa_K808032]] in [[pSB1C3]] ( to design [[BBa_K808032]])  from [[Friday, 17.08.12]] worked well

* 6 colonies are picked in order to perform a [[Colony PCR]] and for inoculation of 5 mL [[DYT-media]]-CAM

* [[Colony PCR]]

: primers: [[VF2]] & [[VR]]

** inoculation of 5 mL [[DYT-media]]-CAM with colony E,F,C,D

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of inoculated 5 mL [[DYT-media]]-CAM with colony E,F,C,D

* [[DNA Digestion]] of 15 µL of preped plasmids with [[EcoRI-HF]] & [[PstI-HF]]

* due to rather bad results of [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] colonies are used for inoculation of 5 mL [[DYT-media]]-CAM again

** [[PhoA]] in [[pSB1C3]], primers [[VF2]] & [[VR]], 1.5 µL each

** [[EstA]] in [[pSB1C3]], primers [[SOE EstA mut up]] & [[BBa EstA down]], 1.5 µL each

* [PCR Clean up]]  

* [[DNA Digestion]] of PCR product with [[EcoRI-HF]] & [[PstI-HF]] over night at 37°C

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] leads to c([[pNB-Est13]]- cut E/P)=25 ng/µL

* [[Ligation]] of digested [[pNB-Est13]] in digested [[pSB1C3]]

* [[Heatshock transformation]] of [[DH5alpha]] with 10 µL of ligation batch

* transformation of ligation from yesterday succeeded, colony picked and inoculation of 5 mL [[DYT-media]]-CAM

** [[BBa_K808032]] (colony C 1.1/5.1 ligated at 4°C), primers [[VF2]], [[XbaI Rbs Phoa up]], [[BBa Ara Promo lo]], [[SE Est13 mut up]], [[SOE Est13 mut lo]], [[BBa EstA down]], [[VR]], 1.5 µL each,  

=== Week 7 / CW 35 ===

** using primers [[VF2]], [[VR]], [[SOE Est13 mut up]], [[SOE Est13 mut lo]]

* test expression of [[BBa_K808032]] (colony C 1.1/5.1 ligated at 4°C) in [[DH5alpha]]

** inoculation of 50 mL [[DYT-media]]-CAM with [[DH5alpha]] carrying [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032]

* positive signal increases with higher temperature, staining succeeded

*  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]

* 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]

* 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]

* 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

* 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

* 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]

* 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

[[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]]

*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/Heat_Shock_Transformation Bacterial transformation by heatshock] of [[BL21(DE3)SHuffle]] with the plasmid pET26b(+)pNBDSM13 encoding Esterase 13 recieved by Henkel AG & Co. KGaA

* bacterial suspension was poured out on LB plates containing [https://2012.igem.org/Team:TU_Darmstadt/Materials/Chloramphenicol Cmp]

*: 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

* [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.

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of [[pEX-Fsc]] in [https://2012.igem.org/Team:TU_Darmstadt/Materials/TOP10 TOP10]

* [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]]

* [[Protein expression of FsC]] at different temperatures (16°C, 25°C, 30°C and 37°C) for the final step of the expression

* 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]

*: Barcode 043 [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX#pEX-FsC pEX-FsC] with primer [[M13 Reverse up]]

*: Barcode 044 [https://2012.igem.org/Team:TU_Darmstadt/Materials/pEX#pEX-FsC pEX-FsC] with primer [[ClaI pIII lo]]

* [[Protein expression of FsC]] according to standard protocol

* [[Protein expression of Est13]] according to standard protocol

*[[Protein expression of FsC]] according to standard protocol

*[[Protein expression of FsC]] according to standard protocol

*[[Protein expression of FsC]] according to standard protocol

[[File:lineburk_fsc.png|center|700px|thumb|Lineweaver-Burk plot of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808025 BBa_K808025]]

[[File:v_abnahme_bei_glykol(2).png|700px|center|thumb| of enzyme activity related to an increasing ehtylenglcol concentration]]

[[File:principle of PET degradation.jpg]]<br />

Conditions: Room temperature (~ 22°C) pH = 7.4

Round about 1g of PET granulate is added in each two falcon tubes to between 15 and 20 mL water of pH = 7.4 and several drops of bromthymol blue. In one of these falcon tubes Est13 stock solution is added until a concentration of 2,5 µM is reached. For 15 days the solutions are incubated at room temperature. At eight of these days the enzymatic solutions and the negative sample are photographed, to control the shift of pH.

[[File:PET degradation Day 1.jpg]]

[[File:PET degradation Day 2.jpg]]

[[File:PET degradation Day 4.jpg]]

[[File:PET degradation Day 7.jpg]]

[[File:PET degradation Day 9.jpg]]

[[File:PET degradation Day 10.jpg]]

[[File:PET degradation Day 13.jpg]]

[[File:PET degradation Day 15.jpg]]<br />

The photos show a decrease of the pH over the time in the sample with Est13. The sample without the enzyme remained unchanged. So the Est13 degrades the PET granulate.