Team:TU Darmstadt/Labjournal/Degradation

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== Degradation ==
== Degradation ==
This page is under heavy construction. Please try not to modify while work in progress.
This page is under heavy construction. Please try not to modify while work in progress.
 +
 +
WORK IN PROGRESS DO NO INTERFERE!!!
 +
 +
== Activity Assays on BBa_K808023 ==
 +
== Activity tests of BBa_K808032 ==
 +
=== Week 1 / KW 35 ===
 +
==== Friday, 31.08.12 ====
 +
* [[activity assay in DYT-media]] with [[DH5alpha]] containing [[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
 +
|}
 +
[[IMAGE]]
 +
* test seemed to have worked: but an induced test tube without PET-granula was missing
 +
=== Week 2 / KW 36 ===
 +
==== Tuesday, 04.09.12 ====
 +
* [[activity assay in DYT-media]] with [[DH5alpha]] containing [[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 !! 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-media]] without bacteria contains CAM, Kan, AMP
 +
[[IMAGE]]
 +
* looks good but test tube 2 shows no significant change of colour
 +
==== Wednesday, 05.09.12 ====
 +
* [[activity assay in DYT-media]] with [[DH5alpha]] containing [[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 !! test tube 5 !! test tube 6 !! test tube 7 !! test tube 8 !! test 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-media]] without bacteria contains CAM, Kan, AMP
 +
[[IMAGE]]
 +
* 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 [[BBA-K808000]] conjugated to GFP
 +
 +
==== Trouble shooting ====
 +
 +
* evaluation shows a very high sensisty of [[BBa_K808000]] even to low concentrations of l-arabinose (expression starts at around 0.01%)
 +
* our lab induced with 1.5% probably killing our [[DH5alpha]] due to extrem expression of our transmembrane construct [[BBa_K808030]]
 +
* starting test expressions with  L-arabinose lower concentrations ranging from 0.05% - 1%
 +
==== Thursday, 06.09.12 ====
 +
* [[activity assay in DYT-media]] with [[DH5alpha]] containing [[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 !! test tube 5 !! test tube 6 !! test tube 7 !! test tube 8 !! test tube 9 !! test tube 10 !! test 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-media]] without bacteria contains CAM, Kan, AMP
 +
[[IMAGE]]
 +
* all induced tubes turned yellow, even without PET-granula as a substrate
 +
 +
=== Week 3 / KW 37 ===
 +
==== Monday, 10.09.12 ====
 +
* [[activity assay in DYT-media]] with [[DH5alpha]] containing [[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 !! test tube 5 !! test tube 6 !! test tube 7 !! test tube 8 !! test tube 9 !! test tube 10 !! test tube 11 !! test tube 12 !! test tube 13 !! test tube 14
 +
|-
 +
| DYT-medium || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL || 5 mL
 +
|-
 +
| PET particle ||no || no || no || no || yes || yes || yes || yes || no || no || no || no || yes || yes
 +
|-
 +
| PET stripe || yes || yes || yes || yes || no || no || no || no || no || no || no || no || no || no
 +
|-
 +
| bacteria || yes || yes || yes || yes || yes || yes || yes || yes || yes || yes || yes || yes || yes || no
 +
|-
 +
| induced || 0.05% L-arabinose || 0.1% L-arabinose || 0.2% L-arabinose || 0.4% L-arabinose || 0.05% L-arabinose || 0.1% L-arabinose || 0.2% L-arabinose || 0.4% L-arabinose|| 0.05% L-arabinose || 0.1% L-arabinose || 0.2% L-arabinose || 0.4% L-arabinose || no || no
 +
|}
 +
* test tube 14: [[DYT-media]] without bacteria contains CAM, Kan, AMP
 +
* 0.05% induced tubes with substrate show difference to tube without substrate
 +
** quantification is possible via meassurement of absorption
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
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* [[activity assay on LB-Tributyrin-CAM-plates]] with L-arabinose concentrations: 0.1%, 0.2%, 0.4%
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
 +
== Protein Expression ==
 +
=== CW 24 ===
 +
==== Thursday, 14.06.2012 ====
 +
* PCR for protein expression of [[FsC]]
 +
** Annotation: each PCR is performed in 5 batches à 50 µL.
 +
** T<sub>A</sub> = 57°C, t<sub>A</sub> = 35 s, t<sub>E</sub> = 65 s
 +
# PCR on [[pEST100]] vector for expression with pEX vector
 +
#: gene of interest: [[FsC]] for designed part [[]]
 +
#: primers: [[pEX FsC His SfiI up]] & [[pEX FsC stop SfiI lo]]
 +
 +
==== Friday, 15.06.2012 ====
 +
*[[AGE]] for quality control [[IMAGE]]
 +
 +
=== CW 25 ===
 +
==== Wednesday, 20.06.2012 ====
 +
*[[AGE]] for preparation [[IMAGE]]
 +
*[[Gel extraction]] of [[PCR]] products
 +
*: Concentration of produced [[FsC]] sequence: 40 ng/µL
 +
 +
==== Thursday, 21.06.2012 ====
 +
*[[DNA digestion]] of [[PCR]] product from 14.06.
 +
** Enzymes used: [[SfiI]]
 +
** NEBuffer: 4
 +
** Digestion time: over night
 +
** Digestion temperature: 50°C
 +
 +
==== Friday, 22.06.2012 ====
 +
*[[Miniprep]] of [[pEX]] vector
 +
*: Concentration range: 240-488 ng/µL
 +
*[[DNA digest]] of [[pEX]]
 +
** Enzymes used: [[SfiI]]
 +
** NEBuffer: 4
 +
** Digestion time: 3 days
 +
** Digestion temperature: 50°C
 +
 +
=== CW 26 ===
 +
==== Monday, 25.06.2012 =====
 +
*[[AGE]] for quality control [[IMAGE]]
 +
*[[Gel extraction]] of [[pEX]] [[IMAGE]] and digested [[FsC]] sequence from 21.06. [[IMAGE]]
 +
*: Concentrations: [[pEX]]: 77 ng/µL, [[FsC]] sequence: 18 ng/µL
 +
* [[Ligation]] of [[pEX]] with [[FsC]] sequence with the ratio 1:3 and 1:5
 +
{| class="wikitable"
 +
|-
 +
! 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 Ligase]] || 1 µL || 1 µL
 +
|-
 +
| H<sub>2</sub>O || 33 µL || 30 µL
 +
|}
 +
* Ligation time: over night
 +
 +
==== Tuesday, 26.06.2012 ====
 +
* [[Bacterial transformation]] by [[electroporation]] of [[Top10]] with the ligation product [[pEX-FsC]] from 25.06.
 +
 +
==== Wednesday, 27.06.2012 ====
 +
* 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 ====
 +
* [[Miniprep]] of [[pEX-Fsc]] in [[Top10]]
 +
*: Concentration: 45 ng/µL and 405 ng/µL
 +
* [[Bacterial transformation]] by [[electroporation]] of [[BmH7118]] with the [[Miniprep]] product [[pEX-FsC]]
 +
 +
=== CW 27 ===
 +
==== Monday, 02.07.2012 ====
 +
* [[Protein expression of FsC]] at different temperatures (16°C, 25°C, 30°C and 37°C) for the final step of the expression
 +
 +
==== Tuesday, 03.07.2012 ====
 +
* [[Purification of Periplasmatic Protein]] for all expression temperatures
 +
 +
==== Wednesday, 04.07.2012 ====
 +
* [[Protein purification]] without cell disrupter
 +
* [[SDS-PAGE (Schägger)]] analysis of all collected samples
 +
{| class="wikitable"
 +
|-
 +
! Expression at 16°C !! Expression at 25°C
 +
|-
 +
| [[IMAGE]] || [[IMAGE]]
 +
|-
 +
! Expression at 30°C !! Expression at 37°C
 +
|-
 +
| [[IMAGE]] || [[IMAGE]]
 +
|}
 +
* The best results were maintained at an expression temperature of 30°C
 +
 +
==== Friday, 06.07.2012 ====
 +
* Inoculation of 5 ml [[DYT]] [[cam]] with [[BmH7118]] containing [[pEX-FsC]]
 +
* Incubation for 3 days at 20°C
 +
 +
=== CW 28 ===
 +
==== Monday, 09.07.2012 ====
 +
* [[Miniprep]] of [[pEX-FsC]] from 06.07.
 +
*: Concentration: 470 ng/µL
 +
* Preparation for sequencing at [[Eurofins]]
 +
*: Barcode 043 [[pEX-FsC]] with primer [[M13 Reverse up]]
 +
*: Barcode 044 [[pEX-FsC]] with primer [[ClaI pIII lo]]
 +
* Plasmid DNA: 5µL
 +
* Primer: 3µL
 +
* H<sub>2</sub>O: 7µL
 +
 +
==== Wednesday, 11.07.2012 ====
 +
* [[Protein expression of FsC]] according to standard protocol
 +
 +
==== Thursday, 12.07.2012 ====
 +
* [[Protein purification]] by standard protocol
 +
* [[SDS-PAGE (Schägger)]] analysis of samples [[IMAGE]]
 +
 +
=== CW 29 ===
 +
==== Monday, 16.07.2012 ====
 +
* [[Protein expression of FsC]] according to standard protocol
 +
 +
==== Tuesday, 17.07.2012 ====
 +
* [[Protein purification]] by standard protocol
 +
* [[SDS-PAGE (Schägger)]] analysis of samples [[IMAGE]]
 +
 +
=== CW 31 ===
 +
==== Monday, 30.07.2012 ====
 +
*[[Protein expression of FsC]] according to standard protocol
 +
 +
==== Tuesday, 31.07.2012 ====
 +
* [[Protein purification]] by standard protocol
 +
* [[SDS-PAGE (Schägger)]] analysis of samples [[IMAGE]]
 +
 +
== SOE PCR ==
 +
=== Week 1 / KW 17 ===
 +
==== Tuesday, 24.04.12 ====
 +
* Production of [[electrocompetent cells]] [[DH5alpha]] and [[BL21]]
 +
* Pouring of [[LB-Agar]] plates with ampecilin resistance (AMP)
 +
* setting of [[DYT media]]
 +
* [[Electroporation]] of [[BL21]] with the following plasmids
 +
** [[pEST100]], carrying a CM-resistance, the genes of [[phoA]], [[FsC]] and [[EstA]]
 +
** [[pET26b(+)]], carrying a Kan-resistance and the gene of [[pNB-Est13]]
 +
** [[pET16]], carrying an Amp-resistance , and is needed for our [[SKV]]
 +
** transformed Bl21 cells are incubated over night at 37°C in DYT-media and crossed out on LB-Agar plates
 +
==== Wednesday, 25.04.12 ====
 +
* [[Miniprep]] of  the 3 overnigth [[Bl21]] cultures and [[concentration meassurement via Nanodrop]]
 +
** [[pEST100]] = 140 ng/µL
 +
** [[pET26b(+)]] = 200 ng/µL
 +
** [[pET16]] = 200 ng/µL
 +
==== Thursday, 26.04.12 ====
 +
* [[pEST100]] and [[pET26b(+)]] serve as templates for the follwoing [[PCR 1]]s
 +
Annotation: Every PCR is done in 4 assays à 50 µL, T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SKV]]
 +
#: primers: [[SKV a1 up XbaI]] & [[SKV a1 lo NdeI]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]]
 +
#: primers: [[SOE A up]] & [[SOE a1 lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[FsC]]
 +
#: primers: [[SOE A up]] & [[SOE a2 lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[FsC]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[EstA part1]]
 +
#: primers: [[SOE b2 up]] & [[SOE b2 lo]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[pNB-Est13 part2]]
 +
#: primers: [[SOE b1 up]] & [[SOE Est13 mut lo]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]]
 +
#: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]]
 +
* [[AGE]] (Agorese gele elektrophoresis) for qualitiy control
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
=== Friday, 27.04.12 ===
 +
* clean up of 1. PCR on [[pEST100]] from [[Thursday, 26.04.12]] via [[Ammonium acetate - Ehtanol DNA precipitation]], solved in 54 µl ddH<sub>2</sub>O
 +
* restriction of cleaned up 1. PCR and [[pET16b]] with XbaI and NdeI over weekend at 37°C
 +
** Annotation: From now on [[SKV]] will be protocolled in [[SKV]] wiki.
 +
== Week 2 / KW 18 ==
 +
==== Monday, 30.04.12 ====
 +
* [[SOE PCR]]
 +
** [[pNB-Est13 part1]] & [[pNB-Est13 part2]], primers: [[SOE b1 up]] & [[SOE b1 lo]]
 +
** [[Promo-LacO-RBS-Phoa]] & [[FsC]], primers: [[SOE A up]] & [[SOE b2 lo]]
 +
** Annotation: Every PCR is done in 4 assays à 50 µL, T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E1</sub> = 20s, t<sub>E1</sub> = 35s
 +
* [[AGE]] of SOE PCR
 +
[[IMAGE]]
 +
* [[SOE PCR]] of [[Promo-LacO-RBS-Phoa-FsC]] worked, [[pNB-Est13]] did not due to missing clean up via [[AGE]], precipitation is insufficient, we do it again
 +
* [[PCR]]s
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[pNB-Est13 part2]]
 +
#: primers: [[SOE b1 up]] & [[SOE Est13 mut lo]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]]
 +
#: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]]
 +
* [[AGE]]
 +
** 1. PCR worked well 2. PCR did not
 +
[[IMAGE]]
 +
==== Wednesday, 02.05.12 ====
 +
* [[PCR]]s
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]]
 +
#: primers: [[SOE A up]] & [[SOE a1 lo]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[pNB-Est13 part2]]
 +
#: primers: [[SOE b1 up]] & [[SOE Est13 mut lo]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]]
 +
#: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]]
 +
: T<sub>A</sub> = 57°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s
 +
: every PCR is performed in 3 batches à 50 µL
 +
* [[AGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
* [[Promega gel extraction]]
 +
** c(1.PCR)=6 ng/µL
 +
** c(2.PCR)=12 ng/µL
 +
** c(3.PCR)=13 ng/µL
 +
* [[SOE PCR]] of 2.PCR and 3.PCR in order to form [[pNB-Est13]] for SOE PCR with [[Promo-LacO-RBS-Phoa]] and [[EstA]]
 +
: T<sub>A1</sub> = 60°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 25 s
 +
: T<sub>A</sub> = 60°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 35 s
 +
==== Tuesday, 03.05.12 ====
 +
* preparative [[AGE]] of SOE PCR from yesterday
 +
* [[Gel extraction]]
 +
** c[[pNB-Est13]] 7 ng/µL
 +
==== Friday, 04.05.12 ====
 +
* [[PCR]]s
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]]
 +
#: primers: [[SOE A up]] & [[SOE a1 lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[pNB-Est13]]
 +
#: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[FsC]]
 +
#: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part2]] for [[SOE PCR]] with [[EstA part1]]
 +
#: primers: [[SOE EstA mut PstI out up]] & [[SOE D lo]]
 +
** annotation: T<sub>A</sub> = 60°C, t<sub>a</sub> = 25 s, t<sub>E</sub> = 35 s
 +
** every PCR is performed in 3 batches à 50 µL
 +
* preparative [[AGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
[[IMAGE]]
 +
=== Week 3 / KW 19 ===
 +
==== Tuesday  08.05.12 ====
 +
* [[SOE PCR]]
 +
: gene of interest: [[Promo-LacO-RBS-Phoa-pNBEst13]] for [[SOE PCR]] with [[EstA]]
 +
: primers: [[SOE A up]] & [[SOE b1 lo]]
 +
: template: [[pNB-Est13]] from last friday & [[Promo-LacO-RBS-Phoa]] from [[Wednesday, 02.05.12]]
 +
** annotation: T<sub>A1</sub> = 60°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 35 s
 +
** T<sub>A2</sub> = 60°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 1 min
 +
* did not work
 +
==== Wednesday, 09.05.12 ====
 +
* [[PCR 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]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] with [[pNB-Est13 part1]] and [[EstA part1]]
 +
#: primers: [[SOE Est13 mut up]] & [[SOE b1 lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[pNB-Est13]]
 +
#: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[FsC]]
 +
#: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part2]] for [[SOE PCR]] with [[EstA part1]]
 +
#: primers: [[SOE EstA mut PstI out up]] & [[SOE D lo]]
 +
** annotation: T<sub>A</sub> = 60°C, t<sub>a</sub> = 25 s, t<sub>E</sub> = 35 s
 +
** every PCR is performed in 2 batches à 50 µL
 +
==== Friday, 09.05.12 ====
 +
* qualitative [[AGE]]
 +
* [[IMAGE]]
 +
* both [[EstA part 1]] (3.PCR & 4.PCR) did not work well
 +
=== Week 4 / KW 20 ===
 +
==== Monday, 14.05.12 ====
 +
* [[Gel extraction]] of remaining PCRs from [[Wednesday, 09.05.12]]
 +
** c([[pNB-Est13 part1]])=24 ng/µL
 +
** c([[pNB-Est13 part2]])=26 ng/µL
 +
** c([[EstA part2]])=13 ng/µL
 +
* new [[SOE EstA mut PstI out lo]] is orderd from Sigma-Aldrich
 +
* [[SOE PCR]]
 +
: gene of interest: [[pNB-Est13]] for [[SOE PCR]] with [[EstA]] and [[Promo-LacO-RBS-Phoa]]
 +
: primers: [[SOE b1 up]] & [[SOE b1 lo]]
 +
: template: [[pNB-Est13 part1]] from last friday & [[pNB-Est13 part2]]
 +
** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 35 s
 +
** T<sub>A2</sub> = 57°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 1.15 min
 +
* [[AGE]]
 +
[[IMAGE]]
 +
==== Tuesday, 15.05.12 ====
 +
* [[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 [[Wednesday, 02.05.12]]
 +
# [[SOE PCR]]
 +
#: gene of interest: [[Promo-LacO-RBS-Phoa-FsC]] for [[SOE PCR]] with [[EstA]]
 +
#: primers: [[SOE A up]] & [[SOE b2 lo]]
 +
#: template: [[FsC]] from [[Thursday, 26.04.12]] & [[Promo-LacO-RBS-Phoa]] from [[Wednesday, 02.05.12]]
 +
** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> = 35 s
 +
** T<sub>A2</sub> = 57°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 1.15 min
 +
** each PCR is performed in 2 batches à 50 µL
 +
==== Wednesday, 16.05.12 ====
 +
* [[SOE PCR]] of [[Promo-LacO-RBS-Phoa-pNBEst13] did not work but [[Promo-LacO-RBS-Phoa-FsC]] worked
 +
* [[Gel extraction]]
 +
** c([[Promo-LacO-RBS-Phoa-FsC]])=10 ng/µL
 +
=== Week 5 / Kw 21 ===
 +
==== Monday, 23.05.12 ====
 +
* [[PCR]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]] and [[EstA part2]]
 +
#: primers: [[SOE c1 up]] & [[SOE EstA mut PstI out lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part1]] for [[SOE PCR]] with [[FsC]] and [[EstA part2]]
 +
#: primers: [[SOE c2 up]] & [[SOE EstA mut PstI out lo]]
 +
==== Tuesday, 22.05.12 ====
 +
* qualitative [[AGE]]
 +
[[IMAGE]]
 +
* [[Gel extraction]]
 +
** c([[EstA part1]] for [[SOE PCR]] with [[FsC]])=4 ng/µL
 +
** c([[EstA part1]] for [[SOE PCR]] with [[pNB-Est13]])=7 ng/µL
 +
==== Wednesday, 23.05.12 ====
 +
* [[SOE PCR]]
 +
# [[SOE PCR]]
 +
#: gene of interest: [[EstA]] for [[SOE PCR]] with [[pNB-Est13]]
 +
#: primers: [[SOE c1 up]] & [[SOE D lo]]
 +
#: template: [[EstA part1]] from yesterday & [[EstA part2]] from [[Monday, 14.05.12]]
 +
# [[SOE PCR]]
 +
#: gene of interest: [[EstA]] for [[SOE PCR]] with [[FsC]]
 +
#: primers: [[SOE c2 up]] & [[SOE D lo]]
 +
#: template: [[EstA part1]] from yesterday & [[EstA part2]] from [[Monday, 14.05.12]]
 +
** annotation: T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> =45 s
 +
** T<sub>A2</sub> = 65°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 90 s
 +
* qualitative [[AGE]]
 +
[[IMAGE]]
 +
* [[Gel extraction]]
 +
** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=42 ng/µL
 +
** c([[EstA]] for [[SOE PCR]] with [[FsC]])=23 ng/µL
 +
==== Thursday, 24.05.12 ====
 +
* [[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 [[FsC]]
 +
#: gene of interest: [[EstA]] for [[SOE PCR]] with [[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: [[FsC]] for [[SOE PCR]] with [[Promo-LacO-RBS-Phoa]] and [[EstA part1]]
 +
#: primers: [[SOE b2 up]] & [[SOE b2 lo]]
 +
** annotation: T<sub>A</sub> = 60°C, t<sub>a</sub> = 25 s, t<sub>E</sub> =45 s
 +
: each PCR is performed in 3 batches à 50 µL
 +
* qualitative [[AGE]]
 +
[[IMAGE]]
 +
** both EstAs worked
 +
** Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13 worked
 +
* [[Gel extraction]]
 +
** c([[EstA]] for [[SOE PCR]] with [[pNB-Est13]])=39 ng/µL
 +
** c([[EstA]] for [[SOE PCR]] with [[FsC]])=20 ng/µL
 +
** c([[Promo-LacO-RBS-Phoa]] for [[SOE PCR]] with [[pNB-Est13]])=13 ng/µL
 +
=== Week 6 / Kw 22 ===
 +
==== Tuesday, 29.05.12 ====
 +
* [[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]]
 +
** annotation: each PCR is performed in 3 batches à 50 µL
 +
** T<sub>A1</sub> = 52°C, t<sub>a1</sub> = 25 s, t<sub>E1</sub> =45 s
 +
** T<sub>A2</sub> = 65°C, t<sub>a2</sub> = 25 s, t<sub>E2</sub> = 90 s
 +
* [[AGE]]
 +
[[IMAGE]]
 +
** [[Promo-LacO-RBS-Phoa-Fsc-EstA]] worked
 +
** [[Promo-LacO-RBS-Phoa-pNBEst13]] worked
 +
==== Thursday, 29.05.12 ====
 +
* [[Gel extraction]]
 +
** c([[Promo-LacO-RBS-Phoa-Fsc-EstA]])= 8 ng/µL
 +
** c([[Promo-LacO-RBS-Phoa-pNBEst13]])= 13 ng/µL
 +
== SKV ==
 +
=== Week 1 / KW 17 ===
 +
==== Tuesday, 24.04.12 ====
 +
* Production of [[electrocompetent cells]] [[DH5alpha]] and [[BL21]]
 +
* Pouring of [[LB-Agar]] plates with ampecilin resistance (AMP)
 +
* setting of [[DYT media]]
 +
* [[Electroporation]] of [[BL21]] with the following plasmids
 +
** [[pEST100]], carrying a CM-resistance, the genes of [[phoA]], [[FsC]] and [[EstA]]
 +
** [[pET26b(+)]], carrying a Kan-resistance and the gene of [[pNB-Est13]]
 +
** [[pET16]], carrying an Amp-resistance , and is needed for our [[SKV]]
 +
==== Wednesday, 25.04.12 ====
 +
* [[Miniprep]] of  the 3 overnigth [[Bl21]] cultures and [[concentration meassurement via Nanodrop]]
 +
** [[pEST100]] = 140 ng/µL
 +
** [[pET26b(+)]] = 200 ng/µL
 +
** [[pET16]] = 200 ng/µL
 +
==== Thursday, 26.04.12 ====
 +
* [[pEST100]] and [[pET26b(+)]] serve as templates for the following [[PCR I]]s
 +
Annotation: Every PCR is done in 4 assays à 50 µL, T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[Promo-LacO-RBS-Phoa]] for [[SKV]]
 +
#: primers: [[SKV a1 up XbaI]] & [[SKV a1 lo NdeI]]
 +
* [[AGE]] (Agarose gele elektrophoresis) for qualitiy control
 +
[[120426 PCR 1-3 siehe Laborbuch 26.4.tif]]
 +
=== Friday, 27.04.12 ===
 +
* clean up of 1. PCR on [[pEST100]] from [[Thursday, 26.04.12]] via [[Ammonium acetate - Ehtanol DNA precipitation]], solved in 54 µl ddH<sub>2</sub>O
 +
* restriction of cleaned up 1. PCR and [[pET16b]] with XbaI and NdeI over weekend at 37°C
 +
== Week 2 / KW 18 ==
 +
=== Monday, 30.04.12 ===
 +
* [[AGE]] of DNA digestion from [[Friday, 27.04.12]]
 +
[[120430 Testrestrikt.XbaINdeI.tif]]
 +
* Only one single band on [[AGE]] image
 +
** For saveness two single digests are performed at 20 µL batches. One with XbaI and the other with NdeI. Incubation for 2 hours.
 +
* [[AGE]] of single digests
 +
[[IMAGE]]
 +
** Enzymes cut once each, so digest should have worked.
 +
* [[Ligation]] of [[PhoA]] in cut (XbaI / NdeI) [[pET16b(+)]]
 +
** Annotation: 30 µL batch, 1:5 ratio, Ligation at 4°C for 2 days till [[Wednesday, 02.05.12]]
 +
== week 4/KW 20 ==
 +
=== Monday, 07.05.===
 +
*[[Electroporation]] of [[DH5alpha]] with 5 µl of the ligation which was performed 2 days before.
 +
* clean up of the PCR-product PhoA [[Friday, 04.05.]]
 +
* [[DNA digestion]] of half of the product with XbaI/NdeI for 1,5h at 37°C
 +
* PhoA is cleaned up by [[AGE]]
 +
* concentration:7,36 ng/µl,10,6 ng/µl
 +
*[[Ligation]] of PhoA x [[pET16b(+)]] for 2h, at the rate of (Vector/Insert) 1:5, 1:3
 +
Annotation: If it does not say anything else, [[Ligation]] is always done in 20µl batches.
 +
=== Tuesday, 08.05. ===
 +
* Transformation of [[DH5alpha]] with [[PhoA]] x [[pET16b(+)]] was not successfull, so it was performed again [[Monday, 07.05.]]
 +
* DNA digestion of [[pET16b(+)]]
 +
** 100 µl are cut with XbaI/NdeI, incubaton-time: 1,5h at 37°C
 +
** product is cleaned up by [[Ammonium acetate - Ehtanol DNA precipitation]]
 +
**concentration: 85 ng/µl
 +
=== Wednesday, 09.05.===
 +
* [[Colony-PCR]]
 +
** gene of interest: [[PhoA]]
 +
** primer:[[SKV a1 up XbaI]], [[SKV a1 lo NdeI]] 
 +
* PhoA was not inserted, but 2 colonies were picked and inoculated in 5 ml [[LB]] Amp medium to look at it again due to a test-restriction.
 +
=== Thursday, 10.05.===
 +
* [[Miniprep]] of [[PhoA]] x [[pET16b(+)]]
 +
* [[DNA digestion]] of the [[Miniprep]]: [[PhoA]] x [[pET16b(+)]]
 +
** enzymes: XbaI/NdeI, incubation: 1,5 h, 37°C
 +
** digest shows that [[PhoA]] was not inserted
 +
** [[Ligation]] is performed again [[Monday, 07.05.]]
 +
* Amplification of the genes [[FSC]], [[Est13]] and [[EstA]] via [[PCR gg 1]]
 +
* templates:
 +
** [[FSC]]: pEST100
 +
** [[Est13]]: pET26b
 +
** [[EstA]]: product of [[SOE-PCR]]
 +
* 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]]
 +
=== Friday, 11.05. ===
 +
* an analytic, 1% agarose-gel proves that the amplification of [[FSC]], [[Est13]] and [[EstA]] was successfull.
 +
[[Image]]
 +
* 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/KW21 ==
 +
=== Monday, 14.05.===
 +
* the [[DNA digestion]] of [[pET16b(+)]] cut with XbaI and NdeI is isolated by [[Ammonium acetate - Ehtanol DNA precipitation]]
 +
* [[LIgation]] of [[phoA]] x [[pET16b(+)]] is used to transform [[DH5alpha]] via [[Electroporation]]
 +
=== Tuesday, 15.05.===
 +
*[[Colony-PCR]]
 +
**primer:
 +
**[[SKV a1 up XbaI]],
 +
**[[SKV a1 lo NdeI]]
 +
*[[IMAGE]]
 +
*PhoA is inserted, so 10 clones are picked to grow them in 5ml DYT over night.
 +
=== Wednesday, 16.05.===
 +
* [[Miniprep]] of 10 clones, the plasmid-DNA is isolated out of 3ml culture per clone.
 +
* [[DNA digestion]] with NdeI and NcoI of the plasmids [[PhoA]] x [[pET16b(+)]] 1-4, so [[FSC]] and [[Est13]] can be added to the construct.
 +
* [[FSC]] is cut with NdeI and NcoI 
 +
** digests are performed in 20µl total volume at 37°C for 1,5h and cleaned up by [[AGE]]
 +
* [[IMAGE]]
 +
* [[PCR1]] of [[EST13]]
 +
** template: product of SOE-PCR, cleaned up by the Promega-Kit
 +
** primer: [[SKV b1 up NdeI]], [[SKV b1 lo NcoI]]
 +
=== Friday, 18.05.===
 +
*[[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]])
 +
** ratio vector/insert: 3:1, 1:1, 1:3
 +
**concentration:
 +
*([[phoA]] x [[pET16b(+)]])= 3,72 ng/µl
 +
*[[FSC]]= 12,22 ng/µl
 +
== week 6/KW 22 ==
 +
=== Monday, 21.05.===
 +
* [[Elektroporation]] of [[DH5alpha]] with the [[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]])
 +
* [[DNA digestion]] of ([[phoA]] x [[pET16b(+)]])(150µl,160µl) for the insertion of [[Est13]]/[[FSC]] and [[Est13]](50µl)
 +
** enzymes: NdeI, NcoI
 +
** incubation: 1,5h, 37°C
 +
** cleaned up via[[AGE]]
 +
** c([[phoA]] x [[pET16b(+)]])= 23,5 ng/µl
 +
** c([[Est13]]= 7,7 ng/µ
 +
=== Tuesday, 22.05.===
 +
* [[Ligation]] of [[Est13]] and ([[phoA]] x [[pET16b(+)]])
 +
** both components have been cut with NdeI and NcoI [[Monday, 21.05.]]
 +
** ratio vector/insert: 1:10, 1:5, 1:3
 +
** incubation: 2h, 25°C
 +
** [[Electroporation]] of dH5alpha using the [[Ligation]] of [[Est13]] x ([[phoA]] x [[pET16b(+)]], 5 µl per 100µl cells
 +
=== Wednesday, 23.05. ===
 +
* [[colony-PCR]]
 +
** aim is to check the insertion of [[Est13]]
 +
** Primer:[[SKV b1 up NdeI]], [[SKV b1 lo NcoI]]
 +
** 8 colonies have been picked and the [[AGE]] proves, that [[Est 13]] has not been inserted into ([[phoA]] x [[pET16b(+)]]
 +
=== Wednesday, 23.05.===
 +
* [[Ligation]] of [[FSC]] and [[Est13]] in ([[phoA]] x [[pET16b(+)]]
 +
** ratio vector/insert: 1:3, 1:5
 +
** c(templates):[[Monday, 21.05.]]
 +
*[[Elektroporation]] of [[DH5alpha]] with [[Est13]] in ([[phoA]] x [[pET16b(+)]]
 +
=== Thursday, 24.05. ===
 +
* [[colony-PCR]] proves, that [[Est13]] has been inserted into ([[phoA]] x [[pET16b(+)]]
 +
** plates are covered with colonies, 8 colonies are chosen for [[colony-PCR]]
 +
** colony No.5 is chosen to be inoculated in 5µ DYT/Amp medium
 +
* [PCR 1] is performed again to amplify [[FSC]] and [[Est13]]
 +
**products are cleaned up by [[AGE]]
 +
**[[IMAGE]]
 +
=== Friday, 25.05. ===
 +
* [[DNA digestion]] of [[FSC]] and [[Est13]] [[Thursday, 24.05.]]
 +
** enzymes: NdeI, NcoI
 +
** incubation: 2days, 37°C
 +
== week 7/KW 23 ==
 +
=== Tuesday, 29.05. ===
 +
* [[DNA digestion]] of [[FSC]] and [[Est13]] is cleaned up via [[AGE]]
 +
* [[MIniprep]] of [[Est13]] x ([[phoA]] x [[pET16b(+)]] via Promega-Kit
 +
* [[Ligation]] of [[FSC]] x ([[phoA]] x [[pET16b(+)]])
 +
** ratio vector/insert: 1:3, 1:5, 1:10
 +
** incubation: 2h, 25°C
 +
* [[Electroporation]] of DH5alpha with 5µl of the [[LIgaton]]: [[FSC]] x ([[phoA]] x [[pET16b(+)]]
 +
* [[PCR 1]] to amplify [[EstA]], (Thursday, 10.05.)
 +
** 4 assays à 50 µL, T<sub>A</sub> = 66°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90s
 +
** cleaned up via Kit
 +
=== Wednesday, 30.05. ===
 +
* [[DNA digestion]] of [[EstA]] (product of PCR, 80 µl) and Plasmid: ([[Est13]] x [[phoA]] x [[pET16b(+)]])
 +
** enzymes: EcoRI, NcoI
 +
** incubation: 1,5h, 37°C
 +
** [[AGE]]
 +
** [[Image]]
 +
** 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.
 +
=== Thursday, 31.05. ===
 +
* [[Miniprep]] of the amplified ([[FSC]] x [[phoA]] x [[pET16b(+)]])
 +
** [[DNA digestion]] to ligate [[EstA]]
 +
** enzymes:NcoI, EcoRI
 +
** incubation: 1,5h, 37°C
 +
* to be sure that the plasmid ([[Est13]] x [[phoA]] x [[pET16b(+)]]) really carries [[Est13]] and [[PhoA]], an analytic [[PCR1]] is performed. Also [[pEt16b(+)]] is used as template with different primers.
 +
* templates:
 +
** [[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]]
 +
*conditions:
 +
** T<sub>A</sub> = 66°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90s
 +
* the [[AGE]] shows, that there is no binding-specifity concerning the genes of interest and the primers, but primers have been checked on mistakes.
 +
* to solve the problem, the [[LIgation]] of [[FSC]] and [[Est13]] shall be repeated from the beginning.
 +
* [[Image]]
 +
* Additionally,the decision was to go on with the Ligation of [[EstA]] into the Plasmids ([[Est13]] x [[phoA]] x [[pET16b(+)]]) and ([[FSC]] x [[phoA]] x [[pET16b(+)]]), in case that the PCR was proceeded under wrong conditions.
 +
=== Friday, 01.06. ===
 +
* [[DNA digestion]] of ([[phoA]] x [[pET16b(+)]])
 +
** enzymes: NcoI, NdeI
 +
** incubation: 1,5 h, 37°C
 +
* [[LIgation]] of [[Est13]] and [[FSC]]x([[PhoA]]x[[pET16b(+)]])
 +
** ratio vector/insert: 1:3, 1:5
 +
** c([[Est13]])= 3 ng/µl
 +
** c([[FSC]])= 11 ng/µl
 +
** c([[[[phoA]] x [[pET16b(+)]])= 29 ng/µl
 +
** incubation: over night, 25°C
 +
* [[DNA digestion]] of ([[FSC]]x ([[PhoA]] x [[pET16b(+)]])
 +
* enzymes: NcoI, EcoRI
 +
* incubation: 1,5h, 37°C
 +
== week 8/KW 24 ==
 +
=== Monday, 04.06. ===
 +
* restriction of  ([[FSC]]x([[PhoA]]x[[pET16b(+)]]) and ([[phoA]]x[[pET16b(+)]]) is cleaned up by [[AGE]]
 +
**[[Image]]
 +
**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]]
 +
=== Tuesday, 05.06. ===
 +
*[[colony-PCR]] on [[EST13]] & [[FSC]] is negative.
 +
=== Wednesday, 06.06. ===
 +
* [[Electroporation]] of [[DH5alpha]] with [[Ligation]]-assays from 01.06.
 +
* [[Ligation]] of [[EstA]][[Wednesday,30.05]] and the Plasmids ([[Est13]]x[[phoA]]x[[pET16b(+)]]),([[FSC]]x[[phoA]]x[[pET16b(+)]]) from [[Monday,04.06.]]
 +
** ratio vector/insert: 1:3,1:5
 +
** incubation: 2h, 25°C
 +
**[[Electroporation]] of [[DH5alpha]] with ([[EstA]]x[[Est13]]x[[phoA]]x[[pET16b(+)]]) and ([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]])
 +
=== Friday, 08.06. ===
 +
* No cells are grown on plates, so the plates are incubated for another two days at RT.
 +
== week8/ KW24 ==
 +
=== Monday, 11.06.===
 +
* all plates are covered with cells, so a [[colony-PCR]] is performed on either [[Est13]],[[FSC]] or [[EstA]]
 +
* primer:
 +
** [[SKV b1 up NdeI]], [[SKV b1 lo NcoI]]
 +
** [[SKV b2 up NdeI]], [[SKV b2 lo NCOI]]
 +
** [[SKV c1 up NcoI]], [[SKV c1 lo EcoRI]]
 +
** Every PCR is done in 8 assays à 50 µL, T<sub>A</sub> = 57°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 90 s
 +
=== Tuesday, 12.06. ===
 +
* [[AGE]] shows, that EstA has been inserted in both Plasmids, ([[Est13]]x[[PhoA]]x[[pET16b(+)]]) and ([[FSC]]x[[PhoA]]x[[pET16b(+)]])
 +
* [[IMAGE]]
 +
* 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.
 +
**[[IMAGE]]
 +
=== Wednesday, 13.06. ===
 +
*[[Miniprep]] of the clones No. 3,4,15 and 16. [[Tuesday, 12.06.]]
 +
** c([[EstA]]x[[FSC]]x[[phoA]]x[[pET16b(+)]])=
 +
** No.3=65,17 ng/µl
 +
** No.4=63,8 ng/µl
 +
** c([[EstA]]x[[Est13]]x[[phoA]]x[[pET16b(+)]])=
 +
** No.15=66,3 ng/µl
 +
** No.16=59,7 ng/µl
 +
* for gene-expression, it is performed a [[Elektroporation]] of [[BL21]]-cells with 1µl of the PLasmid-DNA which has just been isolated.
 +
=== Friday, 15.06.===
 +
* Transformation of [[BL21]] worked well
 +
* [[Tributyrinagar plates]] with 1mM IPTG were generated, to induce the Lac-Promotor of [[pET16b(+)]], and to demonstrate the esterase-activity by the lysis of Tributyrin.
 +
* afterwards [[Tributyrinagar plates]] are inoculated with cells which were transformed by [[Miniprep]] No.3,4,15,16 on [[Wednesday, 13.06.]]
 +
** incubation: 37°C, 2 days
 +
== week9/KW25 ==
 +
=== Monday, 18.06.===
 +
* [[Tributyrinagar plates]] are covered with cells, but no lysis is visible.
 +
** plates are incubated for another day at 25°C.
 +
=== Tuesday, 19.06. ===
 +
* no lysis, plates stay incubated at 25°C.
 +
=== Wednesday, 20.06. ===
 +
* no lysis
 +
=== Friday, 22.06. ===
 +
* The plasmids No.3,4 ([[EstA]]x[[FSC]]x[[PhoA]]x[[pET16b(+)]]) and No.15,16 ([[EstA]]x[[Est13]]x[[PhoA]]x[[pET16b(+)]]) are prepared for sequencing at [[Seqlab]]
 +
* primer:
 +
* ([[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]]
 +
 +
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-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.
== BioBricks ==
== BioBricks ==
-
... 9.9.12 16:00 porting files for wiki ...
+
== BioBricks ==
 +
=== Week 1 / Kw 29 ===
 +
==== Tuesday, 17.07.12 ====
 +
* [[PCR 1]]
 +
: each PCR is performed in 5 batches à 50 µL.
 +
: T<sub>A</sub> = 57°C, t<sub>A</sub> = 30 s, t<sub>E</sub> = 2 min
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[PhoA]] for designing part [[BBa_K808028]]
 +
#: primers: [[BBa PhoA up]] & [[BBa PhoA down]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part1]] for designing part [[BBa_K808026]] via [[SOE PCR]]
 +
#: primers: [[BBa Est13 up]] & [[SOE Est13 mut lo]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part2]] for designing part [[BBa_K808026]] via [[SOE PCR]]
 +
#: primers: [[BBa Est13 down]] & [[SOE Est13 mut up]]
 +
* [[AGE]] for quality control
 +
* preparative [[AGE]]
 +
* [[Gel extraction]] of [[PCR]] products
 +
** 1. PCR (gene of interest: [[phoA]]): c(PhoA)=19 ng/µL
 +
** 2. PCR (gene of interest: [[pNB-Est13 part1]]): c(pNB-Est13 part1)=39 ng/µL
 +
** 3. PCR (gene of interest: [[pNB-Est13 part2]]): c(pNB-Est13 part2)=40 ng/µL
 +
* [[SOE PCR]]
 +
: gene of interest: [[pNB-Est13]] for designing part [[BBa_K808028]]
 +
: primers: [[BBa Est13 up]] & [[BBa Est13 down]]
 +
 
 +
** Annotation: SOE PCR is performed in 5 batches à 50 µL
 +
** T<sub>A1</sub> = 52°C, t<sub>A1</sub> = 25 s, t<sub>E1</sub> = 75 s
 +
** T<sub>A2</sub> = 57°C, t<sub>A2</sub> = 30 s, t<sub>E2</sub> = 2 min
 +
** stored over night at 10°C
 +
 
 +
==== Wednesday, 18.07.12 ====
 +
* qualitative [[AGE]] of [[SOE PCR]] (from [[Tuesday, 17.07.12]])
 +
[[120718 SOE PCR Est13.jpg]]
 +
* preperative [[AGE]]
 +
* [[Gel extraction]] of SOE PCR produkt
 +
* [[PCR 1]]
 +
: gene of interest: [[EstA]] from [[SKV]] [[Date]] for designing part [[BBa_K808027]]
 +
: primers: [[BBa EstA up]] and [[BBa EstA down]]
 +
** Annotation: PCR is performed in 5 batches à 50 µL
 +
** T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 75 s
 +
** GC Buffer is used
 +
* preparative [[AGE]]
 +
[[120718 EstA mit bba.jpg]]
 +
* [[Gel extraction]] of [[PCR]] product
 +
** gene of interest: [[EstA]]: c(pNB-Est13 part2)=119 ng/µL
 +
** stored in freezer
 +
 
 +
==== Thursday, 19.07.12 ====
 +
* inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([[FsC]])
 +
* [[DNA Digestion]] of the following parts
 +
## [[PhoA]] from [[Tuesday, 17.07.12]]
 +
## [[pNB-Est13]] 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
 +
** Digestion time: 2 h
 +
* adding of [[Dephosphatase Antarctica]] from NEB on digested [[pSB1C3]] 1 & 2
 +
* [[Ligation]] into digested and 5' dephosphorylated [[pSB1C3]] 1
 +
** [[pNB-Est13]]
 +
** [[PhoA]]
 +
** [[EstA]]
 +
* [[bacterial transformation]] by [[electroporation]] of [[DH5alpha]]
 +
 
 +
==== Friday, 20.07.12 ====
 +
* the following [[electroporation]] from [[Thursday, 19.07.12]] worked
 +
**[[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 [[FsC]] is already on plate, but to controle it, 2 [[colony PCR]]s are performed
 +
* [[Ligation]] of [[EstA]] into digested and 5' dephosphorylated [[pSB1C3]]
 +
* [[electroporation]] of [[DH5alpha]]
 +
* [[Miniprep]] of inoculated FsC in pSB1C3 ([[BBa_K808025]]) from [[Thursday, 19.07.12]] failed
 +
* inoculation of 5 mL [[DYT-media]] with 5 µL CAM and [[DH5alpha]] carrying pSB1C3 with part [[BBa_K808025]] ([[FsC]])
 +
** incubation at 37°C over weekend of transformations and picked colonies
 +
=== Week 2 / KW 30 ===
 +
==== Monday, 23.07.12 ====
 +
* [[Colony PCR]] of picked colonies from [[Friday, 20.07.12]]
 +
[[120723 BBaPhoA BBa PhoA BBa FsC BBa FsC BBa Est13 BBa Est13 BBa Est13.jpg]]
 +
* 2-3 [[PhoA]] colonies, 4-5 [[FsC]] colonies, 6-8 [[pNB-Est13]] colonies
 +
* [[Miniprep]] of one inoculated colony in DYT-media with CAM from [[Friday, 20.07.12]]
 +
** 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 [[FsC]]
 +
* second transformation of [[EstA]] from [[Friday, 20.07.12]] worked, but too many colonies!
 +
** purity plate is done
 +
==== Tuesday, 24.07.12 ====
 +
* [[Miniprep]] of the 2 [[FsC]] [[DYT-media]]-CAM cultures with [[pSB1C3]] carrying [[FsC]] from yesterday
 +
** c([[FsC]] in [[pSB1C3]])=168 ng/µL
 +
* purity plate of [[EstA]] in [[pSB1c3]] is positive
 +
** colonies picked
 +
** [[Colony PCR]] with positive control by amplfifying an [[SKV]]-[[EstA]]
 +
** Annotation: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min, done with NEB- Phusion so its similiar to [[PCR 1]]
 +
** GC Buffer is used
 +
==== Wednesday, 25.07.12 ====
 +
* [[AGE]] of [[Colony PCR]]
 +
** worked out well
 +
[[120725 Colony BBaEstA positiv probe auf SKV EstA.jpg]]
 +
* 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
 +
==== Thursday, 26.07.12 ====
 +
* Sequencing is ordered. The following premixes are used :
 +
** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VR]], 9 µL ddH<sub>2</sub>O
 +
** 10 µL of c([[PhoA]] in [[pSB1C3]])=220 ng/µL, 1µL [[VF2]], 9 µL ddH<sub>2</sub>O
 +
** 7 µL of c([[FsC]] in [[pSB1C3]])=168 ng/µL, 1µL [[VR]], 7 µL ddH<sub>2</sub>O
 +
** 7 µL of c([[FsC]] in [[pSB1C3]])=168 ng/µL, 1µL [[VF2]], 7 µL ddH<sub>2</sub>O
 +
** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VR]], 9 µL ddH<sub>2</sub>O
 +
** 5 µL of c([[EstA]] in [[pSB1C3]])=350 ng/µL, 1µL [[VF2]], 9 µL ddH<sub>2</sub>O
 +
** 5 µL of c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL, 1µL [[VR]], 9 µL ddH<sub>2</sub>O
 +
** 5 µL of c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL, 1µL [[VF2]], 9 µL ddH<sub>2</sub>O
 +
* 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]]
 +
** [[FsC]] in [[pSB1C3]]
 +
** [[EstA]] in [[pSB1C3]]
 +
** Annotation: incubation is done at 37°C over night
 +
==== Friday, 27.07.12 ====
 +
* inoculated [[pNB-Est13]] in [[pSB1C3]] from [[Thursday, 26.07.12]] has not grown over night
 +
** new colony pcked from plate and incubated in 5 mL [[DYT-media]]-CAM at 37°C over night
 +
* [[Miniprep]] of 3 mL from the following over night [[DYT-media]] cultures from [[Thursday, 26.07.12]]
 +
** c([[PhoA]] in [[pSB1C3]])=440 ng/µL
 +
** c([[pNB-Est13]] in [[pSB1C3]])=450 ng/µL
 +
** c([[FsC]] in [[pSB1C3]])=90 ng/µL
 +
 
 +
=== Week 3 / KW 31 ===
 +
==== Monday, 30.07.12 ====
 +
* [[bacterial transformation]] by [[electroporation]] of [[DH5alpha]] with c([[pNB-Est13]] in [[pSB1C3]])=50 ng/µL from [[Monday, 23.07.12]]
 +
==== Tuesday, 31.07.12 ====
 +
* no results in sequencing our BioBricks / parts from [[Thursday, 26.07.12]] except for [[FsC]] in [[pSB1C3]]
 +
* new [[Colony PCR]](similar to [[PCR 2]]) is done on
 +
** [[PhoA]] in [[pSB1C3]]
 +
** [[FsC]] in [[pSB1C3]]
 +
** [[EstA]] in [[pSB1C3]]
 +
** Annotation: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min
 +
** 1 batch à 50 µL per part
 +
* no signal on qualitative [[AGE]]
 +
** Trouble shooting (see discussion below)
 +
 
 +
==== Trouble shooting ====
 +
 
 +
* Symptoms
 +
** very much transformants on crossed out plates after [[bacterial transformation]] by [[electroporation]]
 +
** at first [[Colony PCR]] is positive
 +
** [[Miniprep]]s result in very high yields (exceeding >200 ng/µL)
 +
** second [[Colony PCR]] is negative
 +
** sequencing failed
 +
** BUT [[FsC]] in [[pSB1C3]]
 +
*** has had not very much transformants
 +
*** has had a seuqencing result
 +
*** its [[Miniprep]]s resulted in medium concentrations, settling around 90 ng/µL
 +
 
 +
* Diagnosis:
 +
** our [[bacterial transformation]] is done by [[electroporation]]
 +
** therefor we use electroporation cuvettes, stored in DNA precipitating liquids like isopropanol or ethanol after being washed out.
 +
** long before we started these electroporation cuvettes were in use.
 +
** if not washed out carefully, DNA debris (like vectors and Inserts) accumulates in these storage liquids, due to precipitation
 +
** when an [[electroporation]] is performed with one of these cuvettes, probably containing only little DNA debris, the cells get transformed with numbers of different precipitated plasmids
 +
** these plasmids contain a variety of resistances, reaching from CAM over KAN to Amp, allowing even "wrong" transformants to grow on selective media
 +
** after a short period of time, bacterial colonies start to seperate and repel their plasmids, selecting only the most "comfortables"
 +
** probably our plasmids, containing big genes on ligated vectors, are pushed out during this phase of selection
 +
** this slow decrease of plasmid could explain the missing second positive signal of [[Colony PCR]]
 +
 
 +
* Strategy
 +
** switching our method from [[Electroporation]] to [[Heatshock transformation]]
 +
 
 +
 
 +
==== Wednesday, 01.08.12 ====
 +
* due to change of strategy the following [[PCR 1]]s are performed
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[PhoA]] BioBrick for assembly of part [[BBa_K808028]]
 +
#: primers: [[BBa PhoA up]] & [[BBa PhoA down]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part1]] for [[SOE PCR]] of EstA ([[BBa_K808027]])
 +
#: primers: [[BBa Est A up]] & [[SOE EstA mut PstI out lo]]
 +
# PCR on [[pEST100]]
 +
#: gene of interest: [[EstA part2]] for [[SOE PCR]] of EstA ([[BBa_K808027]])
 +
#: primers: [[BBa EstA mut up]] & [[BBa EstA down]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part1]] for [[SOE PCR]] of pNB-Est13 ([[BBa_K808026]])
 +
#: primers: [[BBa Est13 up]] & [[BBa Est13 mut lo]]
 +
# PCR on [[pET26b(+)]]
 +
#: gene of interest: [[pNB-Est13 part2]] for [[SOE PCR]] of pNB-Est13 ([[BBa_K808026]])
 +
#: primers: [[BBa Est13 lo]] & [[BBa Est13 mut up]]
 +
** Annotation: every [[PCR]] is performed in 3 batches à 50 µL
 +
** T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 75 s
 +
* due to time pressure there will be no qualitative but only preperative [[AGE]]
 +
** [[PhoA]] only on analytical scale but on same [[AGE]] with [[EstA part1]]
 +
[[120801 soe pcr esta1 pcr phoa.jpg]]
 +
* 1 cut out [[EstA part1]], 2 [[PhoA]]
 +
[[120801 soe pcr est131 est132.jpg]]
 +
* 2 cut out [[pNB-Est13 part1]], 3 cut out [[pNB-Est13 part2]]
 +
[[120801 soe pcr esta2.jpg]]
 +
* cut out [[EstA part2]]
 +
* [[Gel extraction]] of [[EstA part1]], [[EstA part2]], [[pNB-Est13 part1]] and [[pNB-Est13 part2]]
 +
** c([[EstA part1]])=19 ng/µL
 +
** c([[EstA part2]])=110 ng/µL
 +
** c([[pNB-Est13 part1]])=45 ng/µL
 +
** c([[pNB-Est13 part2]])=48 ng/µL
 +
* [[SOE PCR]]s
 +
# SOE PCR
 +
#: gene of interest: [[EstA]]
 +
#: primers: [[EstA part1]] & [[EstA part2]]
 +
# SOE PCR
 +
#: gene of interest: [[pNB-Est13]]
 +
#: primers: [[pNB-Est13 part1]] & [[pNB-Est13 part2]]
 +
** as a control a PCR is performed on [[EstA part2]]
 +
** Annotation: SOE PCR is performed in 3 batches à 50 µL
 +
** T<sub>A1</sub> = 52°C, t<sub>A1</sub> = 30 s, t<sub>E1</sub> = 75 s
 +
** T<sub>A2</sub> = 57°C, t<sub>A2</sub> = 30 s, t<sub>E2</sub> = 75 s
 +
** stored over night at 10°C
 +
* qualitative [[AGE]]
 +
[[120801 SOE PCR Est13 EstA EstApart2 kontrolle.jpg]]
 +
* 2 [[pNB-Est13]], 3 [[ESTA]], 4[[EstA part2]]
 +
* [[PCR Clean up]] by Promega Kit of [[PhoA]], [[pNB-Est13]], [[EstA]]
 +
* [[DNA Digestion]] of the following parts
 +
## [[PhoA]]
 +
## [[pNB-Est13]]
 +
## [[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 ====
 +
* [[Ligation]] into digested and 5' dephosphorylated [[pSB1C3]]
 +
** [[pNB-Est13]]
 +
** [[PhoA]]
 +
** [[EstA]]
 +
** Annotation: Ligation is performed in a 20 µL batch
 +
** digested [[pSB1C3]] still availabe from [[Thursday, 19.07.12]]
 +
** 1:5 & 1:10 ratio
 +
** incubation at room temperature for 30 min
 +
* [[Heatschock transformation]] of [[DH5alpha]] with our ligation batches
 +
* incubation over night at 37°C
 +
==== Friday, 03.08.12 ====
 +
* transformation succeeded
 +
** colonies picked to inoculate 5 ml [[DYT-media]]-CM
 +
==== Saturday, 04.08.12 ====
 +
* [[Miniprep]] of picked colonies
 +
** c([[PhoA]] in [[pSB1C3]])=227 ng/µL
 +
** c([[pNB-Est13]] in [[pSB1C3]])=148ng/µL
 +
** c([[EstA]] in [[pSB1C3]])=227 ng/µL
 +
* test [[DNA Digestion]] with EcoRI-HF and PstI-HF
 +
* [[PCR 2]] on preped plasmids with primers [[VR]] & [[VF2]], and the respective BBa primers
 +
: T<sub>A</sub> = 60°C, t<sub>a</sub> = 35s, t<sub>E</sub> = 25 s
 +
** XylH in [[pSB1C3]] serves as a control in digestion and [[PCR 2]]
 +
* qualitative [[AGE]] 1% agarose
 +
[[120804 Verdau PhoA, EstA, Est13, PCR auf EstA, Est 13, PhoA, Kontrolle auf Insert und Vektor.jpg]]
 +
* digestion: 2 [[PhoA]], 3 [[EstA]], 4 [[pNB-Est13]] PCR: 6 [[EstA]] with BBa primers, 7 [[EstA]] with [[VR]] & [[VF2]], 8 [[pNB-Est13]] with BBa primers, 10 [[pNB-Est13]] with [[VR]] & [[VF2]], 11 [[PhoA]] with BBa primers, 12 [[PhoA]] with [[VR]] & [[VF2]]
 +
* tests succeeded
 +
** Sequencing of the following parts
 +
*** c([[PhoA]] in [[pSB1C3]])=227 ng/µL
 +
*** c([[pNB-Est13]] in [[pSB1C3]])=148ng/µL
 +
*** c([[EstA]] in [[pSB1C3]])=227 ng/µL
 +
* Annotation: failure in mixing sequencing premixes
 +
* retransformation of [[DH5alpha]] by [[Heatshock transformation]] with the following plasmids with 1 µL each
 +
** c([[PhoA]] in [[pSB1C3]])=227 ng/µL
 +
** c([[pNB-Est13]] in [[pSB1C3]])=148ng/µL
 +
** c([[EstA]] in [[pSB1C3]])=227 ng/µL
 +
=== Week 4 / Kw 32 ===
 +
Assembly of our composite part RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808030]]) is reached by synthesis (from GENEART) of 2 different gene parts:
 +
* [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]]
 +
* [[BsaI-Myctag-EstA- bba suffix]]
 +
* the RBS will be added by PCR with primers [[XbaI Rbs Phoa up]] (ignoring the bba prefix but adding an XbaI site upstream of the RBS) & [[Est13 Bsa1 lo]], ignoring the bba prefix but adding an XbaI site upstream of the RBS and for the downstream part primers [[EstA Bsa1 up]] and[[BBa EstA down]] are used
 +
* both products can be assembled by using BsaI. An restriction enzyme, with an restriction site in a defined distance to its recognition site
 +
==== Monday, 06.08.12 ====
 +
* [[Heatshock transformation]] of [[DH5alpha]] with 1 µL synthesis products (c=100 ng/µL):
 +
** [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]]
 +
** [[BsaI-Myctag-EstA- bba suffix]]
 +
* [[DNA Digestion]] of 10 µL = 1 µg of both synthesis products
 +
** [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] digested with EcoRI-HF and BsaI-HF in a 50 µL batch
 +
** [[BsaI-Myctag-EstA- bba suffix]] digested with BsaI-HF and PstI-HF in a 50 µL batch
 +
* preparative [[AGE]]
 +
* Failure due to overlooked additional PstI site in GENEART Vectors
 +
* inoculation of 5 ml [[DYT-media]]-CAM with retransformations from [[Saturday, 04.08.12]]
 +
** [[PhoA]] in [[pSB1C3]]
 +
** [[pNB-Est13]] in [[pSB1C3]]
 +
** [[EstA]] in [[pSB1C3]]
 +
* incubation at 37°C over night
 +
==== Tuesday, 07.08.12 ====
 +
* [[Colony PCR]] on 4 colonies of each transformation of synthesis product from [[Monday, 06.08.12]]
 +
** Annotation: PCR is done with house-taq, similar to [[PCR 2]]
 +
** T<sub>A</sub> = 57°C, t<sub>A</sub> = 30 s, t<sub>E</sub> = 90 s
 +
* [[Colony PCR]] succeeded
 +
** from [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] colonies 5, 2, 11 are picked
 +
** from [[BsaI-Myctag-EstA- bba suffix]] colonies 1, 10, 16 are picked
 +
* inoculation of 5 ml [[DYT-media]]-KAN with colonies
 +
* incubation over night at 37°C
 +
* inoculated 5 mL cultures from [[Monday, 06.08.12]] are stored at 4°C
 +
==== Wednesday, 08.08.12 ====
 +
* [[Miniprep]] of following 5 mL cultures
 +
** from [[Monday, 06.08.12]]
 +
*** c([[PhoA]] in [[pSB1C3]])=90 ng/µL
 +
*** c([[pNB-Est13]] in [[pSB1C3]])=221 ng/µL
 +
*** c([[EstA]] in [[pSB1C3]])=202 ng/µL
 +
** from [[Tuesday, 07.08.12]]
 +
***[[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]]
 +
**** colony 2 c=438 ng/µL
 +
**** colony 5 c=509 ng/µL
 +
**** colony 11 c=291 ng/µL
 +
*** [[BsaI-Myctag-EstA- bba suffix]]
 +
**** colony 1 c=381 ng/µL
 +
**** colony 10 c=420 ng/µL
 +
**** colony 16 c=263 ng/µL
 +
* [[DNA Digestion]]
 +
** of [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] colony 11
 +
*** performed in a 40 µL batch
 +
*** BsaI and EcoRI are used
 +
*** digestion for 3 h
 +
** of [[BsaI-Myctag-EstA- bba suffix]]
 +
*** performed in a 40 µL batch
 +
*** BsaI and SpeI are used
 +
*** digestion for 3 h
 +
* preperative [[AGE]]
 +
[[120808 Restriktion mit E BsaI PhoA Est13 und bsai s EstA.jpg]]
 +
* 3-4 cut out [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]], 5-6 cut out [[BsaI-Myctag-EstA- bba suffix]]
 +
* [[Gel extraction]]
 +
** c([[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]])= 8 ng/µL
 +
** c([[BsaI-Myctag-EstA- bba suffix]])= 26 ng/µL
 +
* [[ligation]] of both inserts in [[pSB1C3]] 2 from [[Thursday, 19.07.12]]
 +
** 1:5:5 ratio is performed in two 20 µL batches (one incubated at 4°C, the other at room temperature over night)
 +
* new sequencing is ordered of the following plasmids
 +
** [[PhoA]] in [[pSB1C3]]
 +
** [[pNB-Est13]] in [[pSB1C3]]
 +
** [[EstA]] in [[pSB1C3]]
 +
*** general premix:
 +
**** 5 µL plasmid
 +
**** primers [[VF2]] & [[VR]], 1.5 µL each
 +
**** 9 µL ddH<sub>2</sub>O
 +
==== Thursday, 09.08.12 ====
 +
* [[Heatshock transformation]]  of [[DH5alpha]] with 10 µL of each ligation batch from yesterday
 +
==== Friday, 10.08.12 ====
 +
* transformation from yesterday suceeded
 +
** one colony per plate (1:5:5 & 1:10:10 at room temperature and 1:5:5 & 1:10:10 at room 4°C)is picked for inoculation of 5 mL [[DYT-media]]-CAM
 +
==== Saturday, 11.08.12 ====
 +
* [[Miniprep]] of picked colonies from yesterday
 +
** testing ligation by [[DNA Digestion]] of plasmids with [[EcorI-HF]] & [[PstI-HF]]
 +
* [[Colony PCR]] on picked colonies
 +
** Annotation: T<sub>A</sub> = 55°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 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]]
 +
* 2-5 [[bba prefix-PhoA-His6tag-pNBEst13-Myctag-EstA- bba suffix]] running at round about 3 kb
 +
=== Week 5 / KW 33 ===
 +
==== Monday, 13.08.12 ====
 +
* Preparation for designing part [[BBa_K808032]] which is an arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
 +
* in order to add RBS upstream of PhoA of [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]] the following [[PCR]]s are performed
 +
# [[PCR 1]] on [[bba prefix-PhoA-His6tag-pNBEst13-BsaI site]], as a template the pure synthesis product is used
 +
#:  T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min
 +
#: primers: [[XbaI Rbs Phoa up]] & [[Est13 Bsa1 lo]]
 +
# [[PCR 1]] on [[BsaI-Myctag-EstA- bba suffix]], as a template the pure synthesis product is used
 +
#: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min
 +
#: primers: [[EstA Bsa1 lo]] & [[BBa EstA down]]
 +
# [[PCR 3]] on preped colony (1:5:5 room temperature from [[Friday, 10.08.12]])
 +
#: T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min
 +
#: primers: [[XbaI Rbs Phoa up]] & [[BBa EstA down]]
 +
** Annotation: each [[PCR]] is performed in 4 batches à 50 µL
 +
* 2 qualitative [[AGE]]
 +
[[120813 PCR 1-4 RBSPE 5-8 EXN-Myc-Est 9-12.jpg]]
 +
* 2-5 [[bba prefix-RBS-PhoA-His6tag-pNBEst13-BsaI]], 6-9 [[BsaI-Myctag-EstA-bba suffix]]
 +
[[120813 PCR Q5 auf bba gesamt konstrukt.jpg]]
 +
* 2-4 [[bba prefix-RBS-PhoA-His6tag-pNBEst13-Myctag-EstA-bba suffix]]
 +
* [[PCR]]s succeeded
 +
* [[PCR clean up]] of 1.-3. PCR
 +
* [[DNA Digestion]] of [[pSB1C3]] carrying [[RFP]] part [[????]]
 +
** enzymes used: [[XbaI]] & [[PstI]]
 +
** performed in 100 µL batch
 +
* [[DNA Digestion]] of part [[BBa_K808000]] ([[pSB1C3]] carrying [[Arabinose inducible promotor]])
 +
** enzymes used: [[SpeI]] & [[PstI]]
 +
** performed in 100 µL batch
 +
* preparative [[AGE]]
 +
** c(1.PCR)= 147 ng/µL
 +
** c(2.PCR)= 133 ng/µL
 +
** c(3.PCR)= 96 ng/µL
 +
** c(cut S/P [[BBa_K808000]]: [[Arabinose inducible promotor]]) possibility 1= 14 ng/µL
 +
** c(cut S/P [[BBa_K808000]]: [[Arabinose inducible promotor]]) possibility 2= 14 ng/µL
 +
** c(cut X/P [[pSB1C3]] carrying [[RFP]])=20 ng/µL
 +
* [[Ligation]] at 4°C over night of the following combinations:
 +
# 1.PCR & 2.PCR in [[pSB1C3]] carrying [[RFP]]
 +
# 3.PCR in [[pSB1C3]] carrying [[RFP]]
 +
# 1.PCR & 2. PCR in [[BBa_K808000]] possibility 1
 +
# 3.PCR in [[BBa_K808000]] possibility 1
 +
# 1.PCR & 2. PCR in [[BBa_K808000]] possibility 2
 +
# 3.PCR in [[BBa_K808000]] possibility 2
 +
==== Tuesday, 14.08.12 ====
 +
* [[Heatshock transformation]] of ligation batches 1-6 from yesterday
 +
==== Wednesday, 15.08.12 ====
 +
* transformation succeeded
 +
* inoculation of 5 mL [[DYT-media]]-CAM of the following colonies
 +
** colony 1.1
 +
** colony 2.1
 +
** colony 2.2
 +
** colony 3.1
 +
** colony 4.1
 +
** colony 4.2
 +
** colony 5.1
 +
** colony 5.2
 +
** colony 6.1
 +
** colony 6.2
 +
*** incubation over night at 37°C
 +
* [[Colony PCR]] on colonies 1.1-6.2 with house-taq
 +
** primers: [[VR]] & [[VF2]]
 +
==== Thursday 16.08.12 ====
 +
* [[Miniprep]] of colonies from yesterday
 +
* [[DNA Digestion]] of preped colonies with [[EcorI-HF]] & [[PstI-HF]]
 +
** incubation for 1.5 h at 37°C
 +
* qualitative [[AGE]]
 +
** FAILURE, because bands on gel are inconsistent
 +
[[120816 Testrestriktion der pSB1C3 mit e und p colony platten 1 - 6.jpg]]
 +
* 2 colony 1.1, 3 colony 2.1, 3 colony 2.2, 4 colony 3.1, 5 colony 4.1, 6 colony 4.2, 7 colony 5.1, 8 colony 5.2
 +
* for further information see discussion below
 +
* new inoculation of 5 mL [[DYT-media]]-CAM of Colonies 1.1 - 5.2
 +
 
 +
==== Trouble shooting ====
 +
 
 +
 
 +
What should have been transformed:
 +
* colony 1.1: 1.PCR & 2.PCR in [[pSB1C3]]
 +
** should be RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808030]]) in [[pSB1C3]] (length: ~ 3.1 kb)
 +
* colony 2.1: 3.PCR in [[pSB1C3]] upstream of part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1
 +
** should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808032]]) in [[pSB1C3]] (length: ~ 4.3kb)
 +
* colony 2.2: similar to colony 2.1
 +
* colony 3.1: 3.PCR in [[pSB1C3]] upstream of part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 2
 +
** should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808032]]) in [[pSB1C3]] (length: ~ 4.3kb) like colony 2.1
 +
* colony 4.1: 3.PCR in [[pSB1C3]]
 +
** should be RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808030]]) in [[pSB1C3]] (length: ~ 3.1 kb)
 +
* colony 4.2: similiar to colony 4.1
 +
* colony 5.1: 1.PCR & 2.PCR in [[pSB1C3]] upstream of part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1
 +
** should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA ([[BBa_K808032]]) in [[pSB1C3]] (length: ~ 4.3kb)
 +
* colony 5.2: similar to colony 5.1
 +
 
 +
Conclusion:
 +
*estimated gene length was aorund 4,3 kb (colony 5.1, 5.2, 2.1, 2.2 = part [[BBa_K808032]]) or 3,1 kb (colony 1.1, 4.1, 4.2 part [[BBa_K808030]])
 +
* digestion with [[EcoRI-HF]] & [[PstI-HF]] of all [[ligations]] from [[Thursday 16.08.12]] in part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1 shows bands at round about 3 - 3.5 kb
 +
* when digested with same enzymes of ligations into part [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 2, bands are shown at round about 1 - 1.5 kb
 +
* this leads to our concluison that [[BBa_K808000]] ([[Arabinose inducible promotor]]) possibility 1 was the not digested DNA band, because resulting gene length of cut insert correlates with length of [[BBa_K808000]] ([[Arabinose inducible promotor]])
 +
* for a sharper solution an [[AGE]] with 0.5 % agarose is performed for digestions of colonies 1.1, 3.1, 4.1, 4.2  plus batches from [[Colony PCR]] from [[Wednesday, 15.08.12]]
 +
[[120816 Colony 1.1 3.1 4.1 4.2 Colony Pcr 1-8.tif]]
 +
* 2-5 colonies 1.1, 3.1, 4.1, 4.2, 6 - 13 colony 1.1, 2.1, 2.2, 3.1, 4.1, 4.2, 5.1, 5.2
 +
 
 +
 
 +
==== Friday, 17.08.12 ====
 +
* the image above means that:
 +
** colony 5.1, 5.2, 2.1, 2.2 are only carrying part [[[[BBa_K808000]] which encodes for the arabinosis inducable promotor
 +
** but colony 1.1, 2.1, 2.2 are carrying 1.PCR & 2.PCR or 3.PCR in [[pSB1C3]]
 +
* [[Miniprep]] of named colonies in liquid DYT-culture from [[Thursday 16.08.12]]
 +
** half culture is preped, half culture serves for [[10% DMSO stocks]]
 +
** c(1.1: [[BBa_K808030]])= 81 ng/µL
 +
** c(4.1: [[BBa_K808030]])= 128 ng/µL
 +
** c(4.2: [[BBa_K808030]])= 79 ng/µL
 +
** c(2.1: [[BBa_K808000]])= 90 ng/µL
 +
** c(2.2: [[BBa_K808000]])= 78 ng/µL
 +
** c(5.1: [[BBa_K808000]])= 64 ng/µL
 +
** c(5.2: [[BBa_K808000]])= 62 ng/µL
 +
* for BioBrick assembly we perform a [[DNA Digestion]] of
 +
** [[BBa_K808000]] (colony 1.1, 4.1, 4.2), with EcoRI / SpeI, serves as upstream part
 +
** [[BBa_K808030]] (colony 5.1, 5.2, 2.1, 2.2), with EcoRI / XbaI, serves as downstream part
 +
* preperative [[AGE]]
 +
* [[Gel extraction]]
 +
** downstream part
 +
** c(1.1: [[BBa_K808030]])= 21.5 ng/µL
 +
** c(4.1: [[BBa_K808030]])= 54 ng/µL
 +
** c(4.2: [[BBa_K808030]])= 58 ng/µL
 +
** upstream part
 +
** c(2.1: [[BBa_K808000]])= 12 ng/µL
 +
** c(2.2: [[BBa_K808000]])= 11 ng/µL
 +
** c(5.1: [[BBa_K808000]])= 10 ng/µL
 +
** c(5.2: [[BBa_K808000]])= 5 ng/µL
 +
* [[ligation]] of upstream infron of downstream part
 +
** colony 5.1 ([[BBa_K808000]]) in colony 1.1 ([[BBa_K808030]])
 +
** colony 2.2 ([[BBa_K808000]]) in colony 4.2 ([[BBa_K808030]])
 +
** colony 2.1 ([[BBa_K808000]]) in colony 4.1 ([[BBa_K808030]])
 +
* inoculation of 5 mL [[DYT-media]]-CAM of the following colonies
 +
** colony 1.1
 +
** colony 2.1
 +
** colony 2.2
 +
** colony 3.1
 +
** colony 4.1
 +
** colony 4.2
 +
** colony 5.1
 +
 
 +
=== Week 6 / KW 34 ===
 +
==== Monday 20.08.12 ====
 +
* [[Miniprep]] of incoucaltes colonies from [[Friday, 17.08.12]]
 +
** c(1.1: [[BBa_K808030]])= 54 ng/µL
 +
** c(4.1: [[BBa_K808030]])= 74 ng/µL
 +
** c(4.2: [[BBa_K808030]])= 60 ng/µL
 +
** c(2.1: [[BBa_K808000]])= 47 ng/µL
 +
** c(2.2: [[BBa_K808000]])= 42 ng/µL
 +
** c(5.1: [[BBa_K808000]])= 56 ng/µL
 +
* sequencing of
 +
** colony 1.1 using [[VR]] & [[VF2]], 1.5 µL each
 +
** colony 5.1 using [[VR]], [[SOE EstA mut lo]], [[SOE EstA mut up]] & [[VF2]], 1.5 µL each
 +
==== Tuesday 21.08.12 ====
 +
* [[Ligation]] of [[BBa_K808000]] upstream of [[BBa_K808032]] in [[pSB1C3]] ( to design [[BBa_K808032]])  from [[Friday, 17.08.12]] worked well
 +
* 6 colonies are picked in order to perform a [[Colony PCR]] and for inoculation of 5 mL [[DYT-media]]-CAM
 +
** colony A 4.1/2.1 ligated at 4°C
 +
** colony B 4.1/2.1 ligated at 37°C
 +
** colony C 1.1/5.1 ligated at 4°C
 +
** colony D 1.1/5.1 ligated at 37°c
 +
** colony E 4.2/2.2 ligated at 4°C
 +
** colony F 4.2/2.2 ligated at 37°C
 +
* [[Colony PCR]]
 +
: T<sub>A</sub> = 55°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 5 min
 +
: primers: [[VF2]] & [[VR]]
 +
* [[AGE]]
 +
** did not work due to immense gene legth ( around 4 kb)
 +
* [[DNA digestion]] for testing is planned
 +
** inoculation of 5 mL [[DYT-media]]-CAM with colony E,F,C,D
 +
==== Wednesday, 22.08.12 ====
 +
* [[Miniprep]] of inoculated 5 mL [[DYT-media]]-CAM with colony E,F,C,D
 +
** c(colony C 1.1/5.1 ligated at 4°C)=46 ng/µL
 +
** c(colony D 1.1/5.1 ligated at 37°c)=46 ng/µL
 +
** c(colony E 4.2/2.2 ligated at 4°C)=60 ng/µL
 +
** c(colony F 4.2/2.2 ligated at 37°C)=58 ng/µL
 +
* [[DNA Digestion]] of 15 µL of preped plasmids with [[EcoRI-HF]] & [[PstI-HF]]
 +
** expected insert length: 4.4 kb ([[BBa_K808000]]+[[BBa_K808030]])
 +
*[[AGE]] with 0.8% agarose
 +
[[Verdau vom Gesamtkonstrukt 4.2_2.2 und 5.1_1.1 beide temperaturen.tif]]
 +
* all bands are in same heights, so ligation worked well
 +
* ligation worked well, bands are in estimated length
 +
* due to rather bad results of [[Miniprep]] colonies are used for inoculation of 5 mL [[DYT-media]]-CAM again
 +
** incubation over night at 37°C
 +
* evaluation of sequencing from [[Wednesday, 08.08.12]]
 +
** PhoA did not work
 +
** EstA is sequenced inconsistent
 +
** pNB-Est13 still got its illegal PstI-site
 +
** but at leat FsC is sequenced completely
 +
* new sequencing of
 +
** [[PhoA]] in [[pSB1C3]], primers [[VF2]] & [[VR]], 1.5 µL each
 +
** [[EstA]] in [[pSB1C3]], primers [[SOE EstA mut up]] & [[BBa EstA down]], 1.5 µL each
 +
* [[PCR 1]] performed on [[BBa_K808032]](preped colony C 1.1/5.1 ligated at 4°C)
 +
** T<sub>A</sub> = 57°C, t<sub>A</sub> = 25 s, t<sub>E</sub> = 2 min
 +
** primers [[BBa Est13 up]] & [[BBa Est13 down]]
 +
** performed in 3 batches à 50 µL
 +
* analytical [[AGE]] 2% agarose
 +
* [PCR Clean up]]
 +
* [[DNA Digestion]] of PCR product with [[EcoRI-HF]] & [[PstI-HF]] over night at 37°C
 +
==== Thursday, 23.09.12 ====
 +
* [[Miniprep]] of colonies from
 +
** c(colony C 1.1/5.1 ligated at 4°C)=147 ng/µL
 +
** c(colony D 1.1/5.1 ligated at 37°c)=131ng/µL
 +
** c(colony E 4.2/2.2 ligated at 4°C)=85 ng/µL
 +
** c(colony F 4.2/2.2 ligated at 37°C)=128 ng/µL
 +
* preparative [[AGE]] of digested [[pNB-Est13]] from yesterday
 +
* [[Gel extraction]] leads to c([[pNB-Est13]]- cut E/P)=25 ng/µL
 +
* [[Ligation]] of digested [[pNB-Est13]] in digested [[pSB1C3]]
 +
** 1:5 ratio is used
 +
* [[Heatshock transformation]] of [[DH5alpha]] with 10 µL of ligation batch
 +
* incubation of transformed cells at 37°C over night
 +
==== Friday, 24.08.12 ====
 +
* transformation of ligation from yesterday succeeded, colony picked and inoculation of 5 mL [[DYT-media]]-CAM
 +
* sequencing of
 +
** [[BBa_K808032]] (colony C 1.1/5.1 ligated at 4°C), primers [[VF2]], [[XbaI Rbs Phoa up]], [[BBa Ara Promo lo]], [[SE Est13 mut up]], [[SOE Est13 mut lo]], [[BBa EstA down]], [[VR]], 1.5 µL each,
 +
=== Week 7 / KW 35 ===
 +
==== Monday, 27.08.12 ====
 +
* [[Miniprep]] of picked colony from [[BBa_K808026]] ([[pNB-Est13]] in [[pSB1C3]])
 +
** c([[pNB-Est13]] in [[pSB1C3]])=116 ng/µL
 +
* sequencing of [[pNB-Est13]] in [[pSB1C3]]
 +
** using primers [[VF2]], [[VR]], [[SOE Est13 mut up]], [[SOE Est13 mut lo]]
 +
* test expression of [[BBa_K808032]] (colony C 1.1/5.1 ligated at 4°C) in [[DH5alpha]]
 +
** [[BBa_K808032]] is an arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
 +
** inoculation of 50 mL [[DYT-media]]-CAM with [[DH5alpha]] carrying [[BBa_K808032]]
 +
*** stop incubation at at OD<sub>600</sub>=0.7
 +
*** storaging cultures on ice for 15mins
 +
*** inducing with ~ 1.5% L-arabinose (using 3.75 mL of 20% mw l-arobinose solution)
 +
*** incubation at 20°C, 25°C and 30°C over night
 +
==== Tuesday, 28.08.12 ====
 +
* [[antibody staining]]
 +
* detection of surface expression of [[BBa_K808032]] by using [[FACS]]
 +
* positive signal increases with higher temperature, staining succeeded
 +
* [[Protein ourification]] of test expression without rinnung IMAC afterwards, just using supernatant and cell debris pellet
 +
** 32 µL of supernatant is used
 +
** 3 mL pellet of each testexpression is treated with more SDS as a detergent, in order to solve membrane proteins
 +
* running two [[SDS-tris gel]]s 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 gel]]s
 +
==== 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 gel]]s
 +
** pellet is treated with n-Dodecyl-ß-maltoside (DDM) in order to solve membrane proteins
 +
[[IMAGE]]
 +
* 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

Revision as of 18:44, 16 September 2012

Contents

Degradation

This page is under heavy construction. Please try not to modify while work in progress.

WORK IN PROGRESS DO NO INTERFERE!!!

Activity Assays on BBa_K808023

Activity tests of BBa_K808032

Week 1 / KW 35

Friday, 31.08.12

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

IMAGE

  • test seemed to have worked: but an induced test tube without PET-granula was missing

Week 2 / KW 36

Tuesday, 04.09.12

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-media without bacteria contains CAM, Kan, AMP

IMAGE

  • looks good but test tube 2 shows no significant change of colour

Wednesday, 05.09.12

Component test tube 1 test tube 2 test tube 3 test tube 4 test tube 5 test tube 6 test tube 7 test tube 8 test 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-media without bacteria contains CAM, Kan, AMP

IMAGE

  • 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 BBA-K808000 conjugated to GFP

Trouble shooting

  • evaluation shows a very high sensisty of BBa_K808000 even to low concentrations of l-arabinose (expression starts at around 0.01%)
  • our lab induced with 1.5% probably killing our DH5alpha due to extrem expression of our transmembrane construct BBa_K808030
  • starting test expressions with L-arabinose lower concentrations ranging from 0.05% - 1%

Thursday, 06.09.12

Component test tube 1 test tube 2 test tube 3 test tube 4 test tube 5 test tube 6 test tube 7 test tube 8 test tube 9 test tube 10 test 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-media without bacteria contains CAM, Kan, AMP

IMAGE

  • all induced tubes turned yellow, even without PET-granula as a substrate

Week 3 / KW 37

Monday, 10.09.12

Component test tube 1 test tube 2 test tube 3 test tube 4 test tube 5 test tube 6 test tube 7 test tube 8 test tube 9 test tube 10 test tube 11 test tube 12 test tube 13 test tube 14
DYT-medium 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL
PET particle no no no no yes yes yes yes no no no no yes yes
PET stripe yes yes yes yes no no no no no no no no no no
bacteria yes yes yes yes yes yes yes yes yes yes yes yes yes no
induced 0.05% L-arabinose 0.1% L-arabinose 0.2% L-arabinose 0.4% L-arabinose 0.05% L-arabinose 0.1% L-arabinose 0.2% L-arabinose 0.4% L-arabinose 0.05% L-arabinose 0.1% L-arabinose 0.2% L-arabinose 0.4% L-arabinose no no
  • test tube 14: DYT-media without bacteria contains CAM, Kan, AMP
  • 0.05% induced tubes with substrate show difference to tube without substrate
    • quantification is possible via meassurement of absorption

IMAGE IMAGE IMAGE

IMAGE IMAGE IMAGE

Protein Expression

CW 24

Thursday, 14.06.2012

  • PCR for protein expression of FsC
    • Annotation: each PCR is performed in 5 batches à 50 µL.
    • TA = 57°C, tA = 35 s, tE = 65 s
  1. PCR on pEST100 vector for expression with pEX vector
    gene of interest: FsC for designed part [[]]
    primers: pEX FsC His SfiI up & pEX FsC stop SfiI lo

Friday, 15.06.2012

CW 25

Wednesday, 20.06.2012

Thursday, 21.06.2012

  • DNA digestion of PCR product from 14.06.
    • Enzymes used: SfiI
    • NEBuffer: 4
    • Digestion time: over night
    • Digestion temperature: 50°C

Friday, 22.06.2012

  • Miniprep of pEX vector
    Concentration range: 240-488 ng/µL
  • DNA digest of pEX
    • Enzymes used: SfiI
    • NEBuffer: 4
    • Digestion time: 3 days
    • Digestion temperature: 50°C

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 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
IMAGE IMAGE
Expression at 30°C Expression at 37°C
IMAGE IMAGE
  • 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

SOE PCR

Week 1 / KW 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
  • AGE (Agorese gele elektrophoresis) for qualitiy control

IMAGE IMAGE IMAGE

Friday, 27.04.12

Week 2 / KW 18

Monday, 30.04.12

IMAGE

  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
  • AGE
    • 1. PCR worked well 2. PCR did not

IMAGE

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

IMAGE IMAGE IMAGE IMAGE

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
    • annotation: TA = 60°C, ta = 25 s, tE = 35 s
    • every PCR is performed in 3 batches à 50 µL
  • preparative AGE

IMAGE IMAGE IMAGE

Week 3 / KW 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 / KW 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
    • annotation: TA1 = 52°C, ta1 = 25 s, tE1 = 35 s
    • TA2 = 57°C, ta2 = 25 s, tE2 = 1.15 min
  • AGE

IMAGE

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

  • qualitative AGE

IMAGE

Wednesday, 23.05.12

  1. SOE PCR
    gene of interest: EstA for SOE PCR with pNB-Est13
    primers: SOE c1 up & SOE D lo
    template: EstA part1 from yesterday & EstA part2 from Monday, 14.05.12
  2. SOE PCR
    gene of interest: EstA for SOE PCR with FsC
    primers: SOE c2 up & SOE D lo
    template: EstA part1 from yesterday & EstA part2 from Monday, 14.05.12
    • annotation: TA1 = 52°C, ta1 = 25 s, tE1 =45 s
    • TA2 = 65°C, ta2 = 25 s, tE2 = 90 s
  • qualitative AGE

IMAGE

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
  • qualitative AGE

IMAGE

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
    • annotation: each PCR is performed in 3 batches à 50 µL
    • TA1 = 52°C, ta1 = 25 s, tE1 =45 s
    • TA2 = 65°C, ta2 = 25 s, tE2 = 90 s
  • AGE

IMAGE

Thursday, 29.05.12

SKV

Week 1 / KW 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
  • AGE (Agarose gele elektrophoresis) for qualitiy control

120426 PCR 1-3 siehe Laborbuch 26.4.tif

Friday, 27.04.12

Week 2 / KW 18

Monday, 30.04.12

120430 Testrestrikt.XbaINdeI.tif

  • Only one single band on AGE image
    • For saveness two single digests are performed at 20 µL batches. One with XbaI and the other with NdeI. Incubation for 2 hours.
  • AGE of single digests

IMAGE

week 4/KW 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.

Image

  • 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/KW21

Monday, 14.05.

Tuesday, 15.05.

Wednesday, 16.05.

Friday, 18.05.

week 6/KW 22

Monday, 21.05.

Tuesday, 22.05.

Wednesday, 23.05.

Wednesday, 23.05.

Thursday, 24.05.

  • colony-PCR proves, that Est13 has been inserted into (phoA x pET16b(+)
    • plates are covered with colonies, 8 colonies are chosen for colony-PCR
    • colony No.5 is chosen to be inoculated in 5µ DYT/Amp medium
  • [PCR 1] is performed again to amplify FSC and Est13

Friday, 25.05.

week 7/KW 23

Tuesday, 29.05.

Wednesday, 30.05.

Thursday, 31.05.

Friday, 01.06.

week 8/KW 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/ KW24

Monday, 11.06.

Tuesday, 12.06.

Wednesday, 13.06.

Friday, 15.06.

week9/KW25

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

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 AGE

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 / KW 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 / KW 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
    • Annotation: every PCR is performed in 3 batches à 50 µL
    • TA = 57°C, tA = 25 s, tE = 75 s
  • due to time pressure there will be no qualitative but only preperative AGE

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 AGE

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
    • XylH in pSB1C3 serves as a control in digestion and PCR 2
  • qualitative AGE 1% agarose

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 / KW 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
    • Annotation: each PCR is performed in 4 batches à 50 µL
  • 2 qualitative AGE

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 / KW 34

Monday 20.08.12

Tuesday 21.08.12

TA = 55°C, tA = 25 s, tE = 5 min
primers: VF2 & VR
  • AGE
    • did not work due to immense gene legth ( around 4 kb)
  • DNA digestion for testing is planned
    • inoculation of 5 mL DYT-media-CAM with colony E,F,C,D

Wednesday, 22.08.12

  • Miniprep of inoculated 5 mL DYT-media-CAM with colony E,F,C,D
    • c(colony C 1.1/5.1 ligated at 4°C)=46 ng/µL
    • c(colony D 1.1/5.1 ligated at 37°c)=46 ng/µL
    • c(colony E 4.2/2.2 ligated at 4°C)=60 ng/µL
    • c(colony F 4.2/2.2 ligated at 37°C)=58 ng/µL
  • DNA Digestion of 15 µL of preped plasmids with EcoRI-HF & PstI-HF
  • AGE with 0.8% agarose

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

  • all bands are in same heights, so ligation worked well
  • ligation worked well, bands are in estimated length
  • due to rather bad results of Miniprep colonies are used for inoculation of 5 mL DYT-media-CAM again
    • incubation over night at 37°C
  • evaluation of sequencing from Wednesday, 08.08.12
    • PhoA did not work
    • EstA is sequenced inconsistent
    • pNB-Est13 still got its illegal PstI-site
    • but at leat FsC is sequenced completely
  • new sequencing of
  • PCR 1 performed on BBa_K808032(preped colony C 1.1/5.1 ligated at 4°C)
  • analytical AGE 2% agarose
  • [PCR Clean up]]
  • DNA Digestion of PCR product with EcoRI-HF & PstI-HF over night at 37°C

Thursday, 23.09.12

  • Miniprep of colonies from
    • c(colony C 1.1/5.1 ligated at 4°C)=147 ng/µL
    • c(colony D 1.1/5.1 ligated at 37°c)=131ng/µL
    • c(colony E 4.2/2.2 ligated at 4°C)=85 ng/µL
    • c(colony F 4.2/2.2 ligated at 37°C)=128 ng/µL
  • preparative AGE of digested pNB-Est13 from yesterday
  • Gel extraction leads to c(pNB-Est13- cut E/P)=25 ng/µL
  • Ligation of digested pNB-Est13 in digested pSB1C3
    • 1:5 ratio is used
  • Heatshock transformation of DH5alpha with 10 µL of ligation batch
  • incubation of transformed cells at 37°C over night

Friday, 24.08.12

Week 7 / KW 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

IMAGE

  • 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
Retrieved from "http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation"