Team:TU Darmstadt/Labjournal/Degradation

<li><a href="/Team:TU_Darmstadt/Human_Practice" title="Human Practice">Human Practice</a><ul>

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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]

# PCR on [[https://2012.igem.org/Team:TU_Darmstadt/Materials/pEST100 pEST100]

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

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

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Gel_extraction Gel extraction] of remaining PCRs from [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Wednesday.2C_09.05.12 Wednesday, 09.05.12]

** c([[pNB-Est13 part1]])=24 ng/µL

** c([[pNB-Est13 part2]])=26 ng/µL

** c([[EstA part2]])=13 ng/µL

* new [[SOE EstA mut PstI out lo]] is orderd from Sigma-Aldrich

* [[SOE PCR]]  

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

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

: template: [[pNB-Est13 part1]] from last friday & [[pNB-Est13 part2]]

* [[Gel extraction]9 of [[SOE PCR]] from yesterday

** c([[pNB-Est13]])=24 ng/µL

* [[SOE PCR]]  

# [[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 [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Wednesday.2C_02.05.12 Wednesday, 02.05.12]

# [[SOE PCR]]

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

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

#: template: [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] from [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Thursday.2C_26.04.12 Thursday, 26.04.12] & [[Promo-LacO-RBS-Phoa]] from [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Wednesday.2C_02.05.12 Wednesday, 02.05.12]

* [[SOE PCR]] of [[Promo-LacO-RBS-Phoa-pNBEst13] did not work but [[Promo-LacO-RBS-Phoa-FsC]] worked

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

** c([[Promo-LacO-RBS-Phoa-FsC]])=10 ng/µL

# PCR on [[pEST100]]

#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]] and [[EstA part2]]

#: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]]

# PCR on [[pEST100]]

#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC] and [[EstA part2]]

#: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]]

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

** c([[EstA part1]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])=4 ng/µL

** c([[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]])=7 ng/µL

* [[SOE PCR]]  

# [[SOE PCR]]

#: 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]]

#: template: [[EstA part1]] from yesterday & [[EstA part2]] from [[Monday, 14.05.12]]

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

** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=42 ng/µL

** c([[EstA]] for [[SOE PCR]] with [https://2012.igem.org/Team:TU_Darmstadt/Materials/FsC FsC])=23 ng/µL

* [[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

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of PhoA in cut (XbaI / NdeI) [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)]

*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of PhoA x [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)] for 2h, at the rate of (Vector/Insert) 1:5, 1:3

* Transformation of [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5alpha] with PhoA x [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)] was not successfull, so it was performed again [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation#Monday.2C_07.05. Monday, 07.05.]

* DNA digestion of pET16b(+)

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Miniprep Miniprep] of PhoA x [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)]

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

* 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  

* DNA digestion of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)] to produce template for a new ligation of PhoA

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of PhoA x pET16b(+) cut with XbaI and NdeI is carried out using different rates of Vector and insert:

* the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)] cut with XbaI and NdeI is isolated by [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Ammonium_sulfate_precipitation Ammonium acetate - Ehtanol DNA precipitation]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of phoA x pET16b(+) is used to transform [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5alpha] via [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Electroporation]

*[[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

*[https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of FSC x (phoA x [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)])

*(phoA x pET16b(+))= 3,72 ng/µl

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Electroporation Elektroporation] of [https://2012.igem.org/Team:TU_Darmstadt/Materials/DH5alpha DH5alpha] with the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/DNA_Ligation Ligation] of FSC x (phoA x [https://2012.igem.org/Team:TU_Darmstadt/Materials/pET16b pET16b(+)])

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of (phoA x pET16b(+))(150µl,160µl) for the insertion of Est13/FSC

** c(phoA x pET16b(+))= 23,5 ng/µl

** c(Est13)= 7,7 ng/µl

* [[Ligation]] of [[Est13]] and ([[phoA]] x [[pET16b(+)]])

** both components have been cut with NdeI and NcoI [[Monday, 21.05.]]

** [[Electroporation]] of dH5alpha using the [[Ligation]] of [[Est13]] x ([[phoA]] x [[pET16b(+)]], 5 µl per 100µl cells

* [[colony-PCR]]

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

** 8 colonies have been picked and the [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Agarose_gel_electrophoresis Agarose gel electrophoresis] proves, that [[Est 13]] has not been inserted into ([[phoA]] x [[pET16b(+)]]

* [[Ligation]] of [[FSC]] and [[Est13]] in ([[phoA]] x [[pET16b(+)]]

** c(templates):[[Monday, 21.05.]]

*[[Elektroporation]] of [[DH5alpha]] with [[Est13]] in ([[phoA]] x [[pET16b(+)]]

* [[colony-PCR]] proves, that [[Est13]] has been inserted into ([[phoA]] x [[pET16b(+))]]

** plates are covered with colonies, 8 colonies are chosen for [[colony-PCR]]

* [PCR 1] is performed again to amplify [[FSC]] and [[Est13]]

* [https://2012.igem.org/Team:TU_Darmstadt/Protocols/Restriction_digest Restriction digest] of [[FSC]] and [[Est13]] [[Thursday, 24.05.]]

=== week 7/CW 23 ===

* [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]