iGEM 2012 — Team Marburg_SYNMIKRO

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# Team:Marburg SYNMIKRO/Notebook

### From 2012.igem.org

* [Home](/Team%3AMarburg_SYNMIKRO)
* [Team](/Team%3AMarburg_SYNMIKRO/Team)
  + [Team members](/Team%3AMarburg_SYNMIKRO/TeamProfile)
  + [Official Team Profile](https://igem.org/Team.cgi?year=2012&team_name=Marburg_SYNMIKRO)
* [Project](/Team%3AMarburg_SYNMIKRO/Project)
  + [Results](/Team%3AMarburg_SYNMIKRO/Results)
* [Notebook](/Team%3AMarburg_SYNMIKRO/Notebook)
  + [Submitted Parts](/Team%3AMarburg_SYNMIKRO/Parts)
  + [Primer List](/Team%3AMarburg_SYNMIKRO/Primer)
* [Human Practice](/Team%3AMarburg_SYNMIKRO/Happening)
  + [Safety](/Team%3AMarburg_SYNMIKRO/Safety)
* [Attributions](/Team%3AMarburg_SYNMIKRO/Attributions)

 [![Main Page](https://static.igem.org/mediawiki/2012/a/ac/Syn_MR_Logo3.png)](https://2012.igem.org/Main_Page)

|  |
| --- |
| Contents   * [1 Week 1](#Week_1)   + [1.1 09.07.2012](#09.07.2012)   + [1.2 10.07.2012](#10.07.2012)   + [1.3 11.07.2012](#11.07.2012)   + [1.4 12.07.2012](#12.07.2012) * [2 Week 2](#Week_2)   + [2.1 16.07.2012](#16.07.2012)   + [2.2 17.07.2012](#17.07.2012)   + [2.3 18.07.2012](#18.07.2012)   + [2.4 19.07.2012](#19.07.2012)   + [2.5 20.07.2012](#20.07.2012) * [3 Week 3](#Week_3)   + [3.1 26.07.2012](#26.07.2012)   + [3.2 27.07.2012](#27.07.2012) * [4 Week 4](#Week_4)   + [4.1 30.07.2012](#30.07.2012)   + [4.2 31.07.2012](#31.07.2012)   + [4.3 01.08.2012](#01.08.2012)   + [4.4 02.08.2012](#02.08.2012)   + [4.5 03.08.2012](#03.08.2012) * [5 Week 5](#Week_5)   + [5.1 06.08.2012](#06.08.2012)   + [5.2 07.08.2012](#07.08.2012)   + [5.3 08.08.2012](#08.08.2012)   + [5.4 09.08.2012](#09.08.2012) * [6 Week 6](#Week_6)   + [6.1 13.08.12](#13.08.12)   + [6.2 14.08.12](#14.08.12)   + [6.3 15.08.2012](#15.08.2012)   + [6.4 16.08.2012](#16.08.2012)   + [6.5 17.08.2012](#17.08.2012) * [7 Week 7](#Week_7)   + [7.1 20.08.2012](#20.08.2012)   + [7.2 21.08.2012](#21.08.2012)   + [7.3 22.08.2012](#22.08.2012)   + [7.4 23.08.2012](#23.08.2012)   + [7.5 24.08.2012](#24.08.2012) * [8 Week 8](#Week_8)   + [8.1 27.08.2012](#27.08.2012)   + [8.2 28.08.2012](#28.08.2012)   + [8.3 29.08.2012](#29.08.2012)   + [8.4 30.08.2012](#30.08.2012)   + [8.5 31.08.2012](#31.08.2012) * [9 Week 9](#Week_9)   + [9.1 03.09.2012](#03.09.2012)   + [9.2 04.09.2012](#04.09.2012)   + [9.3 05.09.2012](#05.09.2012)   + [9.4 06.09.2012](#06.09.2012)   + [9.5 07.09.2012](#07.09.2012) * [10 Week 10](#Week_10)   + [10.1 10.09.2012](#10.09.2012)   + [10.2 11.09.2012](#11.09.2012)   + [10.3 12.09.2012](#12.09.2012)   + [10.4 13.09.2012](#13.09.2012)   + [10.5 14.09.2012](#14.09.2012) * [11 Week 11](#Week_11)   + [11.1 17.09.2012](#17.09.2012)   + [11.2 18.09.2012](#18.09.2012)   + [11.3 19.09.2012](#19.09.2012)   + [11.4 20.09.2012](#20.09.2012)   + [11.5 21.09.2012](#21.09.2012)   + [11.6 22.09.2012](#22.09.2012) |

# Week 1

[all]

09.07. - 15.07.2012

## 09.07.2012

Inoculation
:   *E. coli* strain C600 Mucts62 in dYT-medium

## 10.07.2012

Isolation of chromosomal DNA
:   *E. coli* strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu *c*ts62)

Inoculation
:   *E. coli* strain DH5α and TOP10 in dYT-medium

## 11.07.2012

Preparation of chemically competent *E. coli* cells
:   TOP10

## 12.07.2012

Transformation
:   GFP, GFP\_fusion, CFP, mRFP, RBS, P108, P100, Plac with *E.coli* TOP10

| Name | Biobrick | Plate | Well | Backbone | Resistance |
| --- | --- | --- | --- | --- | --- |
| GFP | [http://partsregistry.org/Part:BBa\_E0040 BBa\_E0040] | 1 | 14K | pSB1A2 | Ampicillin |
| GFP\_fusion | [http://partsregistry.org/Part:BBa\_K125500 BBa\_K125500] | 3 | 2P | pSB1A2 | Ampicillin |
| CFP | [http://partsregistry.org/Part:BBa\_E0020 BBa\_E0020] | 1 | 6A | pSB1A2 | Ampicillin |
| mRFP | [http://partsregistry.org/Part:BBa\_E1010 BBa\_E1010] | 1 | 18F | pSB2K3 | Kanamycin |
| RBS | [http://partsregistry.org/Part:BBa\_J61100 BBa\_J61100] | 1 | 5J | pSB1A2 | Ampicillin |
| P108 | [http://partsregistry.org/Part:BBa\_J23108 BBa\_J23108] | 2 | 2E | BBa\_J61002 | Ampicillin |
| P100 | [http://partsregistry.org/Part:BBa\_J23100 BBa\_J23100] | 1 | 18C | BBa\_J61002 | Ampicillin |
| PLac | [http://partsregistry.org/Part:BBa\_I14032 BBa\_I14032] | 2 | 11P | pSB2K3 | Kanamycin |

# Week 2

16.07. - 22.07.2012

## 16.07.2012

PCR
:   AmiC (primer: [MS1](/Team%3AMarburg_SYNMIKRO/Primer#MS1 "Team:Marburg SYNMIKRO/Primer") + [MS2](/Team%3AMarburg_SYNMIKRO/Primer#MS2 "Team:Marburg SYNMIKRO/Primer"))
:   HU (primer: [MS3](/Team%3AMarburg_SYNMIKRO/Primer#MS3 "Team:Marburg SYNMIKRO/Primer") + [MS4](/Team%3AMarburg_SYNMIKRO/Primer#MS4 "Team:Marburg SYNMIKRO/Primer"))
:   GroES (primer: [MS5](/Team%3AMarburg_SYNMIKRO/Primer#MS5 "Team:Marburg SYNMIKRO/Primer") + [MS6](/Team%3AMarburg_SYNMIKRO/Primer#MS6 "Team:Marburg SYNMIKRO/Primer"))
:   GixR (primer: [MS7](/Team%3AMarburg_SYNMIKRO/Primer#MS7 "Team:Marburg SYNMIKRO/Primer") + [MS8](/Team%3AMarburg_SYNMIKRO/Primer#MS8 "Team:Marburg SYNMIKRO/Primer"))
:   Enhancer (primer: [MS9](/Team%3AMarburg_SYNMIKRO/Primer#MS9 "Team:Marburg SYNMIKRO/Primer") + [MS10](/Team%3AMarburg_SYNMIKRO/Primer#MS10 "Team:Marburg SYNMIKRO/Primer"))
:   ⇒ template-DNA: chromosomal DNA, denaturation: 30 sec at 95°C; annealing: 30 sec at 60°C; elongation: 30 sec at 72°C; 35 cycles

Inoculation
:   GFP
:   GFP\_fusion
:   CFP
:   mRFP
:   RBS
:   P108
:   LacIQ
:   P100

## 17.07.2012

Plasmid preparation
:   GFP
:   GFP\_fusion
:   CFP
:   mRFP
:   RBS
:   P108
:   LacIq
:   P100

## 18.07.2012

1% Agarose gel

[![](/wiki/images/7/70/Syn_MR_Gel1.jpg)](/File%3ASyn_MR_Gel1.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel1.jpg "Enlarge")

M:1kb gene ruler plus, 1:enhancer, 2:GroES, 3:AmiC, 4:HU, 5:GixR, 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP, 10:RBS, 11:LacIQ, 12:P108, 13:P100, 14:λ-DNA (PCR 18.07.2012)

DNA Gel extraction
:   Enhancer
:   GroES
:   HU

GixR Hybridization

* 5 µl primer [MS7](/Team%3AMarburg_SYNMIKRO/Primer#MS7 "Team:Marburg SYNMIKRO/Primer") (100 µM) + 5 µl primer [MS8](/Team%3AMarburg_SYNMIKRO/Primer#MS8 "Team:Marburg SYNMIKRO/Primer") (100 µM)
  + 5 min 99°C
  + over night, room temperature
  + -80°C

## 19.07.2012

Fill-in reaction (primer extension)
:   Enhancer

Restriction
:   Enhancer (EcoRI/PstI)

PCR
:   AmiC (primer: [MS1](/Team%3AMarburg_SYNMIKRO/Primer#MS1 "Team:Marburg SYNMIKRO/Primer") + [MS2](/Team%3AMarburg_SYNMIKRO/Primer#MS2 "Team:Marburg SYNMIKRO/Primer"))

:   ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 85 °C, elongation: 36 sec; 72 °C, 35 cycles, 1) 2 µl DMSO; 1 µl chrom. DNA | 2) 2 µl DMSO; 5 µl chrom. DNA | 3) without DMSO; 1 µl chrom. DNA | 4) without DMSO; 5 µl chrom. DNA | 5) 2 µl DMSO; without chrom. DNA

Test restriction digest
:   7) CFP (EcoRI/PstI)
:   8) GFP (EcoRI/PstI)
:   9) GFP-fusion (EcoRI/PstI)
:   10) mRFP (EcoRI/PstI)
:   ⇒ all negative

1% Agarose gel

[![](/wiki/images/a/ac/Syn_MR_Gel2.jpg)](/File%3ASyn_MR_Gel2.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel2.jpg "Enlarge")

M:1kb gene ruler plus, 1-5:AmiC PCR conditions (see above), 6-9: control digest (E/P), 6:GFP, 7:GFP-Fusion, 8:CFP, 9:mRFP (PCR 19.07.2012)

Control digest
:   CFP (EcoRI/PstI)
:   GFP (EcoRI/PstI)
:   GFP-fusion (EcoRI/PstI)
:   mRFP (EcoRI/PstI)
:   ⇒ restriction over night

## 20.07.2012

1% Agarose gel from the control digest

[![](/wiki/images/f/fc/Syn_MR_Gel3.jpg)](/File%3ASyn_MR_Gel3.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel3.jpg "Enlarge")

M:1kb gene ruler plus, 1:GFP, 2: GFP-Fusion, 3: CFP, 4: mRFP (control digest 18.07.2012)

# Week 3

23.07. - 29.07.2012

## 26.07.2012

Restriction
:   pSB1T3 (XbaI/SpeI and DpnI)
:   pSB1A3 (XbaI/SpeI and DpnI)
:   pSB1K3 (XbaI/SpeI and DpnI)
:   pSB1C3 (XbaI/SpeI and DpnI)

Ligation
:   GixR + pSB1A3
:   Enhancer + pSB1K3
:   cyclisation of pSB1C3, pSB1K3, pSB1A3, pSB1T3 (couldn’t work)

## 27.07.2012

Our lab-dragon "Hugo" is born

[![Syn MR Hugo.jpg](/wiki/images/e/e7/Syn_MR_Hugo.jpg)](/File%3ASyn_MR_Hugo.jpg)

# Week 4

30.07. - 05.08.2012

## 30.07.2012

PCR
:   CFP (primer: [MS16](/Team%3AMarburg_SYNMIKRO/Primer#MS16 "Team:Marburg SYNMIKRO/Primer") + [MS17](/Team%3AMarburg_SYNMIKRO/Primer#MS17 "Team:Marburg SYNMIKRO/Primer")) ⇒ template-DNA: [http://partsregistry.org/Part:BBa\_E0020 BBa\_E0020]
:   ⇒ positive
:   mRFP (primer: [MS14](/Team%3AMarburg_SYNMIKRO/Primer#MS14 "Team:Marburg SYNMIKRO/Primer") + [MS15](/Team%3AMarburg_SYNMIKRO/Primer#MS15 "Team:Marburg SYNMIKRO/Primer")) ⇒ template-DNA: [http://partsregistry.org/Part:BBa\_E1010 BBa\_E1010]
:   ⇒ positive
:   Gin (primer: [MS11](/Team%3AMarburg_SYNMIKRO/Primer#MS11 "Team:Marburg SYNMIKRO/Primer") + [MS13](/Team%3AMarburg_SYNMIKRO/Primer#MS13 "Team:Marburg SYNMIKRO/Primer")) ⇒ template-DNA: chromosomal DNA (that was wrong)
:   RBS-Gin (primer: [MS13](/Team%3AMarburg_SYNMIKRO/Primer#MS13 "Team:Marburg SYNMIKRO/Primer") + [MS12](/Team%3AMarburg_SYNMIKRO/Primer#MS12 "Team:Marburg SYNMIKRO/Primer")) ⇒ template-DNA: chromosomal DNA (that was wrong)
:   ⇒ annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles

Transformation
:   all vector-backbone-ligations
:   Enhancer (K3)
:   GixR (A3)

## 31.07.2012

Result
:   transformation
:   pSB1A3: 3 colonies
:   pSB1K3: 60 colonies
:   pSB1C3: 9 colonies
:   pSB1T3: 0 colonies
:   Enhancer (K3): 0 colonies
:   GixR (A3): 0 colonies

1% Agarose gel

[![](/wiki/images/6/65/Syn_MR_Gel4.jpg)](/File%3ASyn_MR_Gel4.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel4.jpg "Enlarge")

M:Marker, 1: Gix, 2-6: AmiC (PCR 16.07.2012)

1% Agarose gel

[![](/wiki/images/d/d2/Syn_MR_Gel5.jpg)](/File%3ASyn_MR_Gel5.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel5.jpg "Enlarge")

M:1kb gene ruler plus, 1: AmiC(PCR 16.07.2012)

1% Agarose gel

[![](/wiki/images/8/82/Syn_MR_Gel6.jpg)](/File%3ASyn_MR_Gel6.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel6.jpg "Enlarge")

M:1kb gene ruler plus, 1: RBS-Gin from 30.07, 2:AmiC from 16.07., 3:Gin from 30.07., 4:CFP from 30.07., 5:mRFP from 30.07., 6: control (PCR 30.07.2012)

Resuspension of a biobrick
:   [http://partsregistry.org/Part:BBa\_B0010 BBa\_B0010](Terminator)

DNA Gel extraction
:   CFP
:   mRFP

PCR
:   AmiC (primer: [MS1](/Team%3AMarburg_SYNMIKRO/Primer#MS1 "Team:Marburg SYNMIKRO/Primer") + [MS2](/Team%3AMarburg_SYNMIKRO/Primer#MS2 "Team:Marburg SYNMIKRO/Primer"))
:   RBS-Gin (primer: [MS12](/Team%3AMarburg_SYNMIKRO/Primer#MS12 "Team:Marburg SYNMIKRO/Primer") + [MS13](/Team%3AMarburg_SYNMIKRO/Primer#MS13 "Team:Marburg SYNMIKRO/Primer"))
:   Gin (primer: [MS11](/Team%3AMarburg_SYNMIKRO/Primer#MS11 "Team:Marburg SYNMIKRO/Primer") + [MS13](/Team%3AMarburg_SYNMIKRO/Primer#MS13 "Team:Marburg SYNMIKRO/Primer"))
:   ⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 40 sec; 72 °C, 35 cycles

Restriction
:   pSB1A3 (EcoRI/PstI)
:   CFP (EcoRI/PstI)
:   mRFP (EcoRI/PstI)

Transformation
:   pSB1A3
:   pSB1C3
:   pSB1T3
:   Terminator
:   GixR
:   Enhancer

Inoculation
:   pSB1A3
:   pSB1C3
:   pSB1K3

Ligation
:   pSB1A3 + CFP
:   pSB1A3 + mRFP

GixR Hybridization

* 5 µl primer [MS7](/Team%3AMarburg_SYNMIKRO/Primer#MS7 "Team:Marburg SYNMIKRO/Primer") (100 µM) + 5 µl primer [MS8](/Team%3AMarburg_SYNMIKRO/Primer#MS8 "Team:Marburg SYNMIKRO/Primer") (100 µM)
  + 5 min 99°C
  + over night, room temperature
  + -80°C

DNA Gel extraction
:   AmiC

Ligation
:   AmiC + pSV2 (suicide vector)

## 01.08.2012

Result
:   transformation
:   Only BBa\_B0010 colonies present

Plasmid preparation
:   pSB1A3 ⇒ only clone 1, 3 Positive
:   pSB1C3 ⇒ all negative
:   pSB1K3 ⇒ all negative

Ligation
:   GixR hyb. + pJET2\_1
:   ⇒ over blunt ends

Restriction
:   GixR hyb. (EcoRI/PstI)
:   pSB1C3 (EcoRI/PstI)

1% Agarose gel

[![](/wiki/images/b/be/Syn_MR_Gel7.jpg)](/File%3ASyn_MR_Gel7.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel7.jpg "Enlarge")

M:1kb gene ruler plus, 1: RBS-Gin (+DMSO), 2:RBS-Gin (-DMSO), 3:Gin (+DMSO), 4:Gin (-DMSO), 5: control (PCR 31.07.2012)

1% Agarose gel

[![](/wiki/images/5/53/Syn_MR_Gel8.jpg)](/File%3ASyn_MR_Gel8.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel8.jpg "Enlarge")

M:1kb gene ruler plus, 1: AmiC (+DMSO), 2:AmiC (-DMSO), (PCR 31.07.2012)

DNA Gel extraction
:   RBS-Gin
:   Gin
:   AmiC

Test restriction
:   pSB1A3 (EcoRI)

Restriction
:   RBS-Gin (EcoRI/PstI)
:   Gin (EcoRI/PstI)
:   AmiC (EcoRI/PstI)
:   pSB1C3 (EcoRI/PstI)
:   pSB1K3 (EcoRI/PstI)
:   pSB1T3 (EcoRI/PstI)

Ligation
:   cyclisation of pSB1T3

Fill-in reaction (primer extension)
:   Enhancer

Ligation
:   Enhancer and GixR in pSV2
:   Enhancer in pSB1K3
:   GixR in pSB1C3

1% Agarose gel

[![](/wiki/images/0/0f/Syn_MR_Gel9.jpg)](/File%3ASyn_MR_Gel9.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel9.jpg "Enlarge")

M:1kb gene ruler plus, 1-3: pSB1A3, 4-7:pSB1C3, 8-11: pSB1K3 (Control digest 01.08.2012)

:   pSB1A3 c2
:   pSB1C3 c4
:   pSB1K3 c3

Transformation
:   pSB1A3 religation
:   CFP in A3
:   mRFP in A3
:   AmiC in pJET
:   GixR in pJET
:   Enhancer in pJET
:   GixR in C3
:   Enhancer in K3

Ligation
:   AmiC + pSB1T3
:   RBS-Gin + pSB1K3
:   Gin + pSB1K3

## 02.08.2012

Resuspension of a biobrick
:   pSB3C5
:   ⇒ for ccdB

Plasmid preparation
:   BBa\_B0010 (terminator)

Test restriction
:   Terminator c1,2,3,4 (EcoRI/PstI)

2% Agarose gel

[![](/wiki/images/2/2f/Syn_MR_Gel10.jpg)](/File%3ASyn_MR_Gel10.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel10.jpg "Enlarge")

M:1kb gene ruler plus, 1-4: Terminator, M2:Middle Range gene ruler (Control digest 01.08.2012)

Transformation
:   pSB1T3 recycled
:   AmiC in T3
:   Gin-RBS in K3
:   Gin in K3
:   pSB3C5 linear (false)

## 03.08.2012

Plasmid preparation
:   Enhancer in pJET

# Week 5

06.08. - 12.08.2012

## 06.08.2012

Resuspension and transformation of a biobrick
:   BBa\_J04450 in pSB1A3
:   BBa\_J04450 in pSB1C3
:   BBa\_J04450 in pSB1K3
:   BBa\_J04450 in pSB1T3

Restriction
:   GFP-fusion (EcoRI/SpeI)
:   Terminator (XbaI/PstI)
:   Promoter BBa\_J23100 (EcoRI/SpeI)
:   Promoter BBa\_J23108 (EcoRI/SpeI)
:   RBS (XbaI/PstI)

Ligation
:   RBS-Gin in T3
:   Gin in T3
:   GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3
:   Promoter BBa\_J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3
:   Promoter BBa\_J23100 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3

GixR Hybridization

* 1(3) µl primer [MS7](/Team%3AMarburg_SYNMIKRO/Primer#MS7 "Team:Marburg SYNMIKRO/Primer")7 (100 µM) + 1(3) µl primer [MS8](/Team%3AMarburg_SYNMIKRO/Primer#MS8 "Team:Marburg SYNMIKRO/Primer") (100 µM) + T4-Ligasebuffer
  + 5 min 95°C
  + over night, room temperature
  + -80°C

Transformation
:   GixR in C3
:   Gin in T3
:   RBS-Gin in T3
:   mRFP in A3
:   CFP in A3

## 07.08.2012

Transformation
:   GFP-fusion
:   CFP
:   J23108-RBS
:   J23100-RBS

Plasmid preparation
:   GixR 1,2,3

Restriction
:   GixR hyb. 1,2,3 (EcoRI/PstI)
:   pSB1C3 (EcoRI/PstI)

Ligation
:   GixR hyb. 1,2,3 in pJET
:   GixR hyb. 1,2,3 in C3

Transformation
:   GixR hyb. 1,2,3 in pJET
:   GixR hyb. 1,2,3 in C3

Test restriction
:   pSB3C5 (XbaI)
:   pJET (XbaI)

1% Agarose gel

[![](/wiki/images/9/94/Syn_MR_Gel11.jpg)](/File%3ASyn_MR_Gel11.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel11.jpg "Enlarge")

M:1kb gene ruler plus, 1-4: pSB3C5, 5-8:pSB3C5, 9-12: Enhancer-pJET (Control digest 07.08.2012)

:   ⇒ positive: pSB3C5 3,4,7,8,; Enhancer-pJET 9-12

## 08.08.2012

Result transformation
:   GFP-fusion (20 clones)
:   J23108-RBS (20 clones)
:   J23100-RBS (20 clones)
:   GixR hyb. 1,2,3 in pJET (10 clones)
:   GixR hyb. 1,2,3 in C3 (10 clones)
:   CFP (5 clones)

Restriktion
:   Enhancer in pJET (EcoRI/PstI)
:   pSB1C3 (EcoRI/PstI)

2% Agarose gel

[![](/wiki/images/1/15/Syn_MR_Gel12.jpg)](/File%3ASyn_MR_Gel12.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel12.jpg "Enlarge")

M:1kb gene ruler plus, 1: Enhancer, 2:pSB1C3, (Control digest 08.08.2012)

DNA Gel extraction
:   Enhancer
:   pSB1C3

Ligation
:   Enhancer 1,2 in C3

Transformation
:   Enhancer 1,2 in C3
:   C3
:   mRFP

## 09.08.2012

Result transformation
:   GFP-fusion ⇒ positive
:   J23108-RBS ⇒ positive
:   J23100-RBS ⇒ positive
:   CFP ⇒ positive
:   GixR in C3 c1 ⇒ negative
:   GixR in C3 c2 ⇒ negative
:   GixR in C3 c3 ⇒ negative

Plasmid preparation
:   GFP-fusion
:   J23108-RBS
:   J23100-RBS
:   CFP

Test restriction
:   GFP-fusion (EcoRI/PstI)
:   J23108-RBS (EcoRI)
:   J23100-RBS (EcoRI)
:   CFP (EcoRI/PstI)

1% Agarose gel

[![](/wiki/images/2/24/Syn_MR_Gel13.jpg)](/File%3ASyn_MR_Gel13.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel13.jpg "Enlarge")

M:1kb gene ruler plus, 1-18: GFP-Fusion - Terminator, (Control digest 09.08.2012)

1% Agarose gel

[![](/wiki/images/f/fc/Syn_MR_Gel14.jpg)](/File%3ASyn_MR_Gel14.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel14.jpg "Enlarge")

M:1kb gene ruler plus, 1-18: P108-RBS, (Control digest 09.08.2012)

1% Agarose gel

[![](/wiki/images/4/4e/Syn_MR_Gel15.jpg)](/File%3ASyn_MR_Gel15.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel15.jpg "Enlarge")

M:1kb gene ruler plus, 1-18: P100-RBS, (Control digest 09.08.2012)

1% Agarose gel

[![](/wiki/images/2/25/Syn_MR_Gel16.jpg)](/File%3ASyn_MR_Gel16.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel16.jpg "Enlarge")

M:1kb gene ruler plus, 1-2: GFP-Fusion - Terminator, 3-4: P108-RBS, 5-6: P100-RBS, 7-11: CFP (Control digest 09.08.2012)

Inoculation
:   GixR in C3
:   Gin in T3
:   Gin-RBS in T3
:   pSB1C3
:   Enhancer in C3
:   mRFP in A3

# Week 6

13.08. - 19.08.2012

## 13.08.12

PCR
:   GroES (primer: [MS5](/Team%3AMarburg_SYNMIKRO/Primer#MS5 "Team:Marburg SYNMIKRO/Primer") + [MS6b](/Team%3AMarburg_SYNMIKRO/Primer#MS6b "Team:Marburg SYNMIKRO/Primer"))
:   HU (primer: [MS3](/Team%3AMarburg_SYNMIKRO/Primer#MS3 "Team:Marburg SYNMIKRO/Primer") + [MS4b](/Team%3AMarburg_SYNMIKRO/Primer#MS4b "Team:Marburg SYNMIKRO/Primer"))
:   ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles

1% Agarose gel

[![](/wiki/images/d/db/Syn_MR_Gel17.jpg)](/File%3ASyn_MR_Gel17.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel17.jpg "Enlarge")

M:1kb gene ruler plus, 1: HU (+DMSO), 2: HU (-DMSO), 3: GroES (+DMSO), 4: GroES (-DMSO), (PCR 13.08.2012)

Transformation
:   LacIQ (BBa\_I14032)

## 14.08.12

Restriction
:   pSB1K3 (EcoRI/PstI)
:   Enhancer 3,4 (EcoRI/ PstI)
:   GFP-fusion (EcoRI/SpeI)
:   Terminator (XbaI/PstI)

Test restriction
:   RBS-Gin in T3 (EcoRI/ PstI)

Ligation
:   GFP-fusion (EcoRI/SpeI) + Terminator (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
:   Promotor J23108 (EcoRI/SpeI) + RBS (XbaI/PstI) in pSB1K3 (EcoRI/PstI)
:   Enhancer 3,4 (EcoRI/ PstI) + pSB1K3 (EcoRI/PstI)

Transformation
:   GFP-fusion-Terminator in pSB1K3
:   Promotor-RBS in pSB1K3
:   Enhancer 3 in pSB1K3
:   Enhancer 4 in pSB1K3
:   GixR in pJET

## 15.08.2012

PCR
:   GroES (primer: [MS5](/Team%3AMarburg_SYNMIKRO/Primer#MS5 "Team:Marburg SYNMIKRO/Primer") + [MS6b](/Team%3AMarburg_SYNMIKRO/Primer#MS6b "Team:Marburg SYNMIKRO/Primer"))
:   HU (primer: [MS3](/Team%3AMarburg_SYNMIKRO/Primer#MS3 "Team:Marburg SYNMIKRO/Primer") + [MS4b](/Team%3AMarburg_SYNMIKRO/Primer#MS4b "Team:Marburg SYNMIKRO/Primer"))
:   ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C, 35 cycles

1% Agarose gel

[![](/wiki/images/a/a3/Syn_MR_Gel18.jpg)](/File%3ASyn_MR_Gel18.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel18.jpg "Enlarge")

M:1kb gene ruler plus, 1: HU (-DMSO), 2: HU (+DMSO), 3: GroES (-DMSO), 4: GroES (+DMSO), (PCR 15.08.2012)

DNA Gel extraction
:   HU 1,2

Resuspension of a biobrick
:   BBa\_J04450 in pSB1A3
:   BBa\_J04450 in pSB1T3
:   BBa\_J04450 in pSB1C3
:   BBa\_J04450 in pSB1K3

Transformation
:   BBa\_J04450 in pSB1A3
:   BBa\_J04450 in pSB1T3
:   BBa\_J04450 in pSB1C3
:   BBa\_J04450 in pSB1K3

Test restriction
:   pSB1T3 (EcoRI/PstI and DpnI)
:   pSB1K3 (EcoRI/PstI and DpnI)
:   pSB1C3 (EcoRI/PstI and DpnI)
:   pSB1A3 (EcoRI/PstI and DpnI)

## 16.08.2012

PCR
:   GroES (primer: [MS5](/Team%3AMarburg_SYNMIKRO/Primer#MS5 "Team:Marburg SYNMIKRO/Primer") + [MS6b](/Team%3AMarburg_SYNMIKRO/Primer#MS6b "Team:Marburg SYNMIKRO/Primer"))
:   ⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles

1% Agarose gel

[![](/wiki/images/2/2d/Syn_MR_Gel19.jpg)](/File%3ASyn_MR_Gel19.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel19.jpg "Enlarge")

M:1kb gene ruler plus, 1: GroES (-DMSO), 2: GroES (+DMSO), (PCR 16.08.2012)

Plasmid preparation
:   P-RBS

Test restriction
:   P-RBS (EcoRI/PstI)

1% Agarose gel

[![](/wiki/images/4/4f/Syn_MR_Gel20.jpg)](/File%3ASyn_MR_Gel20.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel20.jpg "Enlarge")

M:1kb gene ruler plus, 1: control, 2: P108-RBS, (control digest 16.08.2012)

Restriction
:   Enhancer 3,4 (EcoRI/ PstI)
:   HU (EcoRI/ PstI)
:   AmiC (EcoRI/ PstI)
:   Promoter, GFP (EcoRI/SpeI)
:   RBS; Terminator (XbaI/PstI)

Ligation
:   HU, AmiC in pSB1A3
:   Enhancer 3,4 in pSB1K3
:   P-RBS in pSB1K3
:   GFP fusion-Terminator in pSB1K3

DNA Gel extraction
:   GroES 1, P-RBS

Transformation
:   AmiC
:   HU
:   Enhancer 3,4, 3 alt,4 alt

Inoculation
:   BBa\_J04450 in pSB1A3
:   BBa\_J04450 in pSB1T3
:   BBa\_J04450 in pSB1C3
:   BBa\_J04450 in pSB1K3
:   AmiC
:   P-RBS

## 17.08.2012

Restriction
:   GroES (EcoRI/SpeI)

Ligation
:   GroES in pSB1C3

Transformation
:   GroES in pSB1C3

Test restriction
:   P-RBS (EcoRI/PstI)
:   BBa\_J04450 in pSB1A3 (EcoRI/PstI)
:   BBa\_J04450 in pSB1T3 (EcoRI/PstI)
:   BBa\_J04450 in pSB1C3 (EcoRI/PstI)
:   BBa\_J04450 in pSB1K3 (EcoRI/PstI)

1% Agarose gel

[![](/wiki/images/2/2d/Syn_MR_Gel21.jpg)](/File%3ASyn_MR_Gel21.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel21.jpg "Enlarge")

M:1kb gene ruler plus, 1-4: BBa\_J04450 in pSB1A3, 5-8: BBa\_J04450 in pSB1K3 P108-RBS, 9-12: BBa\_J04450 in pSB1C3, (control digest 17.08.2012)

1% Agarose gel

[![](/wiki/images/f/f9/Syn_MR_Gel22.jpg)](/File%3ASyn_MR_Gel22.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel22.jpg "Enlarge")

M:1kb gene ruler plus, 1-4: P-RBS (E/P), 5-8: P-RBS (RsaI), 9-12: BBa\_J04450 in pSB1T3, (control digest 17.08.2012)

# Week 7

20.08. - 26.08.2012

## 20.08.2012

PCR
:   Backbone (primer [MS24](/Team%3AMarburg_SYNMIKRO/Primer#MS24 "Team:Marburg SYNMIKRO/Primer") + [MS25](/Team%3AMarburg_SYNMIKRO/Primer#MS25 "Team:Marburg SYNMIKRO/Primer"))
:   ⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles

1% Agarose gel

[![](/wiki/images/6/60/Syn_MR_Gel23.jpg)](/File%3ASyn_MR_Gel23.jpg)

[![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel23.jpg "Enlarge")

M:1kb gene ruler plus, 1: pSB1A3, 2: pSB1T3, 3: pSB1C3, 4: pSB1K3 (PCR 20.08.2012)

Transformation
:   BBa\_JH023

Inoculation
:   GroES
:   P-RBS
:   GFP-T
:   Enhancer 3,4
:   AmiC
:   P-RBS alt
:   GFP-T alt

## 21.08.2012

Results
:   colonies of BBa\_JH023
:   inoculations except AmiC and P-RBS alt successful

PCR
:   pSB1A3-BBa\_J04450 (template DNA)
:   pSB1T3-BBa\_J04450 (template DNA)
:   pSB1C3-BBa\_J04450 (template DNA)
:   pSB1K3-BBa\_J04450 (template DNA)
:   used primer --> [MS24](/Team%3AMarburg_SYNMIKRO/Primer#MS24 "Team:Marburg SYNMIKRO/Primer") + [MS25](/Team%3AMarburg_SYNMIKRO/Primer#MS25 "Team:Marburg SYNMIKRO/Primer")
:   initial denaturation 98°C - 5min
:   denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
:   72°C - 5min --> 4°C - 8

miniprep
:   GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old)

Test restriction
:   of miniprep
:   over night, 37°C

1% Agarose gel
:   [![](/wiki/images/f/fb/Syn_MR_Gel24.jpg)](/File%3ASyn_MR_Gel24.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel24.jpg "Enlarge")

    M:1kb gene ruler plus, 1:pSB1A3, 2:pSB1K3, 3:pSB1C3, 4:pSB1T3, 5: negative control, (PCR 21.08.2012)

Inoculation
:   E.coli BBa\_I14032
:   over night, 37°C

## 22.08.2012

PCR
:   CFP ([MS16](/Team%3AMarburg_SYNMIKRO/Primer#MS16 "Team:Marburg SYNMIKRO/Primer") + [MS17](/Team%3AMarburg_SYNMIKRO/Primer#MS17 "Team:Marburg SYNMIKRO/Primer"))
:   mRFP ([MS14](/Team%3AMarburg_SYNMIKRO/Primer#MS14 "Team:Marburg SYNMIKRO/Primer") + [MS15](/Team%3AMarburg_SYNMIKRO/Primer#MS15 "Team:Marburg SYNMIKRO/Primer"))
:   initial denaturation 98°C - 5min
:   denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 35 times
:   72°C - 5min --> 4°C - 8

1% Agarose gel
:   [![](/wiki/images/b/b0/Syn_MR_Gel25.jpg)](/File%3ASyn_MR_Gel25.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel25.jpg "Enlarge")

    M:1kb gene ruler plus, 1:mRFP, 2:mRFP, 3:CFP, 4:CFP (PCR 22.08.2012)

miniprep
:   of LacIQ

Test restriction
:   of LacIQ with EcoRI and PstI

1% Agarose gel
:   [![](/wiki/images/2/24/Syn_MR_Gel26.jpg)](/File%3ASyn_MR_Gel26.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel26.jpg "Enlarge")

    M:1kb gene ruler plus, 1-4:LacIQ, (control digest 22.08.2012)

1% Agarose gel
:   [![](/wiki/images/b/b2/Syn_MR_Gel27.jpg)](/File%3ASyn_MR_Gel27.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel27.jpg "Enlarge")

    M:1kb gene ruler plus, 1-4:GroES, 5-12: GFT-Terminator, (control digest 21.08.2012)

1% Agarose gel
:   [![](/wiki/images/e/ef/Syn_MR_Gel28.jpg)](/File%3ASyn_MR_Gel28.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel28.jpg "Enlarge")

    M:1kb gene ruler plus, 1-12:Enhancer, (control digest 21.08.2012)

1% Agarose gel
:   [![](/wiki/images/7/78/Syn_MR_Gel29.jpg)](/File%3ASyn_MR_Gel29.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel29.jpg "Enlarge")

    M:1kb gene ruler plus, 1-4:Enhancer, 5-12: P-RBS, (control digest 21.08.2012)

:   test restriction of Enhancer (->21.08.12), gel 2%
:   watch picture
:   test restriction of Enhancer and P-RBS (->21.08.12), gel 2%
:   watch picture

## 23.08.2012

PCR
:   pSB1K3-BBa\_J04450 (template DNA)
:   pSB1T3-BBa\_J04450 (template DNA)
:   used primer --> [MS24](/Team%3AMarburg_SYNMIKRO/Primer#MS24 "Team:Marburg SYNMIKRO/Primer") + [MS25](/Team%3AMarburg_SYNMIKRO/Primer#MS25 "Team:Marburg SYNMIKRO/Primer")
:   initial denaturation 98°C - 5min
:   denaturation: 98°C - 1min; annealing: 55°C - 30s; elongation: 72°C - 2.5min -->repeat 35 times
:   72°C - 5min --> 4°C - 8

1% Agarose gel
:   [![](/wiki/images/7/7a/Syn_MR_Gel31.jpg)](/File%3ASyn_MR_Gel31.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel31.jpg "Enlarge")

    M:gene ruler mix ladder, 1-2:pSB1K3-BBa\_J04450, 3-4: pSB1T3-BBa\_J04450, (PCR 23.08.2012)

Restriction
:   GixR (EcoRI/PstI)

Ligation
:   GixR in pSB1C3

Transformation
:   GixR in pSB1C3

## 24.08.2012

PCR
:   pSB1A3-BBa\_J04450 (template DNA)
:   pSB1T3-BBa\_J04450 (template DNA)
:   pSB1C3-BBa\_J04450 (template DNA)
:   pSB1K3-BBa\_J04450 (template DNA)
:   initial denaturation 98°C - 5min
:   denaturation: 98°C - 10s; annealing: 60°C - 30s; elongation: 72°C - 40s -->repeat 30 times
:   72°C - 10min --> 4°C - 8
:   primer: [MS22](/Team%3AMarburg_SYNMIKRO/Primer#MS22 "Team:Marburg SYNMIKRO/Primer") + [MS23](/Team%3AMarburg_SYNMIKRO/Primer#MS23 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/a/a9/Syn_MR_Gel32.jpg)](/File%3ASyn_MR_Gel32.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel32.jpg "Enlarge")

    M:gene ruler mix ladder, 1-2:pSB1A3-BBa\_J04450, 3-4: pSB1K3-BBa\_J04450, 5-6:pSB1C3-BBa\_J04450, 7-8: pSB1T3-BBa\_J04450, (PCR 24.08.2012)

Restriction
:   RBS (XbaI/PstI)
:   CFP (EcoRI/PstI)
:   mRFP (EcoRI/PstI)

Ligation
:   mRFP and CFP in pSB1C3

Transformation
:   mRFP and CFP in pSB1C3

# Week 8

27.08. - 02.09.2012

## 27.08.2012

Results
:   CFP: 1 colony
:   CFP (neu): Colonies
:   mRFP: colonies
:   mRFP (neu): no colonies

PCR
:   template: backbones (pSB1K3/T3/C3/A3)
:   parameters: 98°C 5min, >> 98°C 1min, 70°C 30s, 72°C 2.5min << x30, 72°C 5min, 4°C 8
:   primer: [MS22](/Team%3AMarburg_SYNMIKRO/Primer#MS22 "Team:Marburg SYNMIKRO/Primer") + [MS23](/Team%3AMarburg_SYNMIKRO/Primer#MS23 "Team:Marburg SYNMIKRO/Primer")

colony-PCR
:   parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C 8
:   primer: [MS18](/Team%3AMarburg_SYNMIKRO/Primer#MS18 "Team:Marburg SYNMIKRO/Primer") + [MS19](/Team%3AMarburg_SYNMIKRO/Primer#MS19 "Team:Marburg SYNMIKRO/Primer")

Agarose gel of backbone PCR
:   no result

elution of the backbones of Friday the 24th

1% Agarose gel
:   [![](/wiki/images/0/05/Syn_MR_Gel33.jpg)](/File%3ASyn_MR_Gel33.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel33.jpg "Enlarge")

    M:1kb gene ruler plus, 1-2:gixR, 3-9: LacIQ-RBS-Gin, 10: CFP, (colony PCR 27.08.2012)

1% Agarose gel
:   [![](/wiki/images/e/e3/Syn_MR_Gel34.jpg)](/File%3ASyn_MR_Gel34.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel34.jpg "Enlarge")

    M:1kb gene ruler plus, 1-4:CFP, 5-10: mRFP, (colony PCR 27.08.2012)

Inoculation
:   pSB1C3\_...
:   mRFP clone 20,24
:   CFP clone 14,15,17,18
:   gix clone 1,2
:   5ml LB, over night, 37°C

## 28.08.2012

production of competent TOP10

PCR
:   Enhancer, Terminator, Gix, P-RBS
:   parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
:   primer: [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/c/c8/Syn_MR_Gel35.jpg)](/File%3ASyn_MR_Gel35.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel35.jpg "Enlarge")

    M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9-10: Enhancer, (PCR 28.08.2012)

Transformation
:   [http://partsregistry.org/Part:BBa\_I0500 pBad/araC] (plate:1, Well:N14)
:   pSacB
:   over night, 37°C

miniprep
:   GixR (1,2)
:   CFP (14,15,17,18)
:   mRFP (20,24)
:   alle in pSB1C3

## 29.08.2012

Results
:   Transformation: no colonies

PCR
:   Terminator, Gix, Enhancer, P-RBS
:   diluted templates: Terminator, P-RBS, Gix 1 --> 1:10
:   Enhancer, Gix 2 --> 1:20
:   parameters: 98°C 5', 98°C 1', 66°C 30", 72°C 30", 72°C 5', 4°C ~
:   primer: [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/e/e2/Syn_MR_Gel36.jpg)](/File%3ASyn_MR_Gel36.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel36.jpg "Enlarge")

    M:1kb gene ruler plus, 1:Terminator, 2-3: Enhancer, 4-6: P-RBS, 7-8: GixR, 9: Enhancer control, 10: P-RBS control, 11: GixR control, (PCR 29.08.2012)

3AA
:   CFP, mRFP with Terminator
:   2µl DNA
:   1µl SpeI
:   2µl Tango
:   15µl dH2O
:   --> 1h at 37°C
:   +1µl EcoRI
:   +2,5µl Tange
:   --> 1h at 37°C
:   --> inactivation for 20min at 80°C

Ligation
:   3AA products in pSB1T3
:   18.5µl H2O
:   2.5 µl Buffer
:   1µl Insert CFP or mRFP (EcoRI + SpeI)
:   1µl Insert Terminator (BBa\_B0010) (XbaI + PstI)
:   1µl pSB1T3 (EcoRI + PstI)
:   1µl Ligase
:   --> 1h at RT

Transformation
:   pBad --> TOP10 + pSB2K3
:   CFP-T, mRFP-T --> TOP10 + pSB1T3

Sequencing
:   CFP\_fwd
:   CFP\_rev
:   mRFP\_fwd
:   mRFP\_rev
:   P-RBS\_fwd
:   GixR\_fwd
:   GixR\_fwd
:   Enhancer\_fwd

## 30.08.2012

Results
:   Transformation: no colonies --> testing Trafo with BBa\_J04450 A/K/T/C
:   2 colonies at pBad-Trafo from 28.08.2012 --> liquid culture for miniprep
:   1 colony at pSacB-Trafo from 28.08.2012 --> tested on succrose-sensitivity

## 31.08.2012

PCR
:   SacB
:   Parameters No.1: 95°C 5', >>95°C 30", 60°C 30", 72°C 1'<< x30, 72°C 5', 4°C
:   Parameters No.2: 95°C 5', >>95°C 30", 72°C 1.5'<< x30, 72°C 5', 4°C
:   Primer [MS26](/Team%3AMarburg_SYNMIKRO/Primer#MS26 "Team:Marburg SYNMIKRO/Primer") + [MS27](/Team%3AMarburg_SYNMIKRO/Primer#MS27 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/1/10/Syn_MR_Gel37.jpg)](/File%3ASyn_MR_Gel37.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel37.jpg "Enlarge")

    M:1kb gene ruler plus, 1,3,5:SacB, 4,5: none, (PCR 31.08.2012)

Ligation
:   3AA (CFP-T, mRFP-T)
:   watch 29.08.
:   used vector: pSB1K3/T3

Transformation
:   of ligation in TOP10

miniprep
:   pBad/araC K.1+2

restriction
:   GixR rehybridization
:   cut with EcoRI and PstI

ligation
:   1µl GixRhyb E+P
:   1µl pSB1C3 E+P
:   1µl Th-Pol
:   2µl Buffer
:   15µl dH2O

Transformation
:   ligation + TOP10 --> LB(cam)

Results of test-trafo
:   Amp, Km, Cm OK --> Km with fewer colonies
:   Tet --> hardly colonies
:   --> comp.TOP10 obviously all right, exclusion of Tet in future work

# Week 9

03.09. - 09.09.2012

## 03.09.2012

Results
:   Transformation
:   GixR in C3 --> no colonies
:   CFP-T/mRFP-T in C3(31.8.) --> no colonies
:   CFP-T/mRFP-T in T3(31.8.) --> many but small colonies
:   CFP-T/mRFP-T in T3(29.8.) --> less colonies

colony-PCR
:   of colonies (CFP-T, mRFP-T)
:   parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
:   primer [MS18](/Team%3AMarburg_SYNMIKRO/Primer#MS18 "Team:Marburg SYNMIKRO/Primer") + [MS19](/Team%3AMarburg_SYNMIKRO/Primer#MS19 "Team:Marburg SYNMIKRO/Primer")

Agarose gel
:   6x colony pT3-CFP-T from 29./31.8.
:   6x colony pT3-mRFP-T from 29./31.8.
:   pC3-CFP (14)
:   pC3.mRFP (24)

1% Agarose gel
:   [![](/wiki/images/6/60/Syn_MR_Gel38.jpg)](/File%3ASyn_MR_Gel38.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel38.jpg "Enlarge")

    M:1kb gene ruler plus, 1-6:CFP-Terminator, 7:CFP control, 8-13:mRFP-Terminator 14: mRFP control, 15-16: none, (colony-PCR 03.09.2012)

## 04.09.2012

Results
:   Transformation
:   CFP-T/mRFP-T in T3
:   2 µl -> 5-6 colonies
:   30 µl -> 1-2 colonies
:   Gix R in C3 (3.9) --> 4 colonies

colony-PCR
:   GixR
:   CFP-T
:   mRFP-T
:   parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

gel extraction
:   P108 (E/S)
:   RBS (X/P)

ligation
:   SacB1 + pSB1C3
:   P-RBS + pSB1C3

Transformation
:   ligation + TOP10 --> LB(cmp)

1% Agarose gel
:   [![](/wiki/images/4/43/Syn_MR_Gel40.jpg)](/File%3ASyn_MR_Gel40.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel40.jpg "Enlarge")

    M:1kb gene ruler plus, 1-2:pBAD/araC, (mini-prep 31.08.2012)

Inoculation
:   pBad/araC
:   5ml LB, over night, 37°C

1% Agarose gel
:   [![](/wiki/images/6/67/Syn_MR_Gel41.jpg)](/File%3ASyn_MR_Gel41.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel41.jpg "Enlarge")

    M:1kb gene ruler plus, 1-4:GixR pSB1C3, 5-9:CFP-Terminator, 10:CFP control, (colony-PCR 04.09.2012)

1% Agarose gel
:   [![](/wiki/images/6/64/Syn_MR_Gel42.jpg)](/File%3ASyn_MR_Gel42.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel42.jpg "Enlarge")

    M:1kb gene ruler plus, 1-9:mRFP-Terminator, 10:mRFP control, (colony-PCR 04.09.2012)

## 05.09.2012

Results
:   Transformation
:   SacB in C3
:   P-RBS in C3

colony PCR
:   SacB in C3
:   P-RBS in C3
:   parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

miniprep
:   Gix in C3
:   pBad

Results
:   Transformation
:   CFP-T (03.09) --> 10 colonies

colony PCR
:   P108-RBS, SacB, GixR
:   parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
:   primer [MS19](/Team%3AMarburg_SYNMIKRO/Primer#MS19 "Team:Marburg SYNMIKRO/Primer") + [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/9/98/Syn_MR_Gel43.jpg)](/File%3ASyn_MR_Gel43.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel43.jpg "Enlarge")

    **no marker**, 1:none, 2-7:SacB, 8-11:P108-RBS, (colony-PCR 05.09.2012)

1% Agarose gel
:   [![](/wiki/images/c/c4/Syn_MR_Gel44.jpg)](/File%3ASyn_MR_Gel44.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel44.jpg "Enlarge")

    **no marker**, 1-5:P108-RBS, 6:none, 7-9:GixR, 10:Scar, 11-12: pBad, 13-15: pGixR, (colony-PCR 05.09.2012)

restriction
:   pSB1A3 (E/P)
:   pSB1K3 (E/P)
:   pSB1C3 (E/P)
:   pSB1T3 (E/P)
:   GFP-T K3 (X/P)
:   GFP K3 (X/P)
:   CFP C3 (X/P)
:   mRFP C3 (X/P)
:   AmiC A (E/S)
:   GroES A (E/S)
:   Terminator (X/P)

Inoculation
:   pRBS
:   SacB
:   mRFP-Terminator
:   5ml LB, over night, 37°C

## 06.09.2012

miniprep
:   pRBS
:   SacB
:   mRFP-Terminator

ligation
:   AmiC (E/S) + GFP (X/P) + pSB1T3 (E/P)
:   AmiC (E/S) + RFP (X/P) + pSB1T3 (E/P)
:   AmiC (E/S) + GCFP (X/P) + pSB1T3 (E/P)
:   AmiC (E/S) + Terminator (X+P + pSB1T3 (E/P)
:   GroES (E/S) + GFP (X/P) + pSB1T3 (E/P)
:   GroES (E/S) + RFP (X/P) + pSB1T3 (E/P)
:   GroES (E/S) + CFP (X/P) + pSB1T3 (E/P)
:   GroES (E/S) + Terminator (X/P) + pSB1T3 (E/P)

PCR of miniprep
:   SacB in pSB1C3
:   p-RBS in pSB1C3
:   terminator in pSB1T3
:   parameters: 98°C 5min, >> 98°C 30 sec, 72°C 1,5min << x30, 72°C 5min, 4°C ∞
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/b/b5/Syn_MR_Gel45.jpg)](/File%3ASyn_MR_Gel45.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel45.jpg "Enlarge")

    M:1kb gene ruler plus, 1:Scar, 2-5:P-RBS, 6-9:SacB, 10-16: mRFP, 17: control, (PCR of miniprep (06.09.2012)

transformation
:   RBS-Gin T3
:   amiC A3
:   groES A3
:   groES-Terminator T3
:   groES-GFP T3
:   groES-CFP T3
:   groES-mRFP T3
:   amiC-Terminator T3
:   amiC-GFP T3
:   amiC-CFP T3
:   amiC-mRFP T3

## 07.09.2012

restriction (6.9.)

ligation
:   AmiC in pSB1K3
:   GroES in pSB1K3
:   mRFP in pSB1K3
:   Terminator in pSB1K3
:   CFP in pSB1K3
:   GFP in pSB1K3
:   GFP+Terminator in C3
:   Enhancer in K3

transformation
:   Ligation
:   AmiC
:   GroES
:   RBS-Gin

miniprep
:   CFP-Terminator

Sequencing
:   mRFP T3
:   P-RBS K12
:   P-RBS K15
:   SacB K6
:   SacB K6

# Week 10

10.09. - 16.09.2012

## 10.09.2012

Restriction
:   Gin-RBS (01.08)

Ligation
:   Gin-RBS in pSB1C3

Transformation
:   Gin-RBS in pSB1C3

Hybridisation
:   GixR
:   98 °C 20 min
:   minus 1 °C to 4 °C in 2 min

Ligation and Transformation
:   GixR (X+S) + pSB1K3 (X+S)

Inoculation (Tetracycline)
:   GroES-mRFP
:   GroES-GFP
:   GroES-CFP
:   amiC-mRFP
:   amiC-GFP
:   amiC-CFP
:   5ml LB, over night, 37°C

Inoculation (Kanamycine)
:   GroES-GFP
:   amiC-mRFP
:   amiC-GFP
:   GroES-Terminator
:   Enhancer
:   5ml LB, over night, 37°C

Inoculation (Chloramphenicol)
:   GFP-Terminator
:   5ml LB, over night, 37°C

Inoculation (Ampicillin)
:   Terminator
:   5ml LB, over night, 37°C

## 11.09.2012

microscopy-screening
:   GroES-GFP ⇒; no fluorescence
:   GroES-CFP ⇒; no fluorescence
:   AmiC-mRFP ⇒; no fluorescence
:   AmiC-GFP ⇒; no fluorescence

colony-PCR
:   GixR
:   RBS-Gin
:   Terminator
:   parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

3A-Assembly (digestion, ligation, transformation)
:   GroES (E/S) + Terminator (X/P) + pSB1K3 (P/E)
:   AmiC (E/S) + Terminator (X/P) + pSB1K3 (P/E)

plasmid-prep and control-digestion (E/P)
:   GroES-GFP
:   GroES-CFP
:   AmiC-mRFP
:   AmiC-GFP
:   GroES-Terminator
:   Enhancer
:   GFP-Terminator
:   Terminator

## 12.09.2012

Results
:   Transformation
:   - P-RBS --> colonies
:   - pB10 --> no colonies
:   - RBS (B0030) --> colonies
:   - Gro-ES/ AmiC-Terminator --> no colonies

colony-PCR
:   Gix (2. try)
:   Gin-RBS (2. try)
:   RBS (B0030)
:   P-RBS (108)
:   parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

Agarose gel
:   Gin-RBS (5x)
:   Gin (1x)
:   Gix (5x)
:   RBS (6x)
:   P-RBS (6x)

1% Agarose gel
:   [![](/wiki/images/1/1f/Syn_MR_Gel46.jpg)](/File%3ASyn_MR_Gel46.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel46.jpg "Enlarge")

    M:1kb gene ruler plus, 1:Scar, 2-6:RBS-Gin, 7:Gin control 8-12: gixR, 13: control, (colony-PCR 12.09.2012)

1% Agarose gel
:   [![](/wiki/images/5/5f/Syn_MR_Gel47.jpg)](/File%3ASyn_MR_Gel47.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel47.jpg "Enlarge")

    M:1kb gene ruler plus, 1:Scar, 2-6:RBS BBa\_B0030, 7-12:P-RBS, 13: control, (colony-PCR 12.09.2012)

Restriction (miniprep 11.09.12)
:   gr-GFP
:   gr-CFP
:   gr-T K3
:   enhancer K3
:   am-RFP-K3
:   am-GFP-K3
:   GFP-T-C3
:   terminator A2

1% Agarose gel
:   [![](/wiki/images/2/2b/Syn_MR_Gel48.jpg)](/File%3ASyn_MR_Gel48.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel48.jpg "Enlarge")

    M:1kb gene ruler plus, 1-3:GroES-GFP, 4:GroES-GFP control, 5:GroES-CFP, 6:GroES-CFP control, 7-10: GroES-Terminator, 11: GroES-Terminator control, 12-15: Enhancer, 16: Enhancer control, (control digest 12.09.2012)

1% Agarose gel
:   [![](/wiki/images/8/8c/Syn_MR_Gel49.jpg)](/File%3ASyn_MR_Gel49.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel49.jpg "Enlarge")

    M:1kb gene ruler plus, 1-2:AmiC-mRFP, 3:AmiC-mRFP control, 4-5:AmiC-GFP, 6:AmiC-GFP control, 7-11: GFP-Terminator, 12: GFP-Terminator control, 13-15: Terminator, 16: Terminator control, (control digest 12.09.2012)

Transformation
:   groES-T

    :   2 Transformations:

        :   - One with a 1:10 dilution of Terminator from ligation,
        :   - another one with a 1:100 dilution of Terminator from ligation
:   amiC-T

    :   2 Transformations:

        :   - One with a 1:10 dilution of Terminator from ligation,
        :   - another one with a 1:100 dilution of Terminator from ligation

PCR
:   Gin-RBS

    :   3 reactions:

        :   -One using High Fidelity (HF) Reaction Buffer,
        :   -one using GC-Buffer
        :   -and another one using self-made reaction buffer
:   Primer used : [MS12](/Team%3AMarburg_SYNMIKRO/Primer#MS12 "Team:Marburg SYNMIKRO/Primer") + [MS13](/Team%3AMarburg_SYNMIKRO/Primer#MS13 "Team:Marburg SYNMIKRO/Primer")
:   Template DNA : *E. coli* C600 chromosomal DNA

## 13.09.2012

Results
:   Transformation

    :   - pBAD (11.09.12) --> no colonies
    :   - AmiC-T (11.09.12) --> 2 x 2 colonies
    :   - GroES-T (11.12.09)--> 2 colonies

colony-PCR
:   AmiC-T & GroES-T (transformation 11.09.12)
:   parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/3/32/Syn_MR_Gel50.jpg)](/File%3ASyn_MR_Gel50.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel50.jpg "Enlarge")

    M:1kb gene ruler plus, 1:RBS-Gin HF, 2:RBS-Gin GC, 3:RBS-Gin SM (PCR 13.09.2012)

Gel extraction
:   RBS-Gin (HF) --> 51 ng/µl
:   RBS-Gin (GC) --> 69 ng/µl
:   RBS-Gin (SM) --> 56 ng/µl

Ligation
:   GroES-T & AmiC-T in pSB1K3

Restriction (preparative digest)
:   GroES (A module)

    :   with SpeI
:   AmiC (A module)

    :   with SpeI
:   mRFP-C3 (B module)

    :   with EcoRI and XbaI
:   CFP-C3 (B module)

    :   with EcoRI and XbaI
:   Gin-RBS

    :   HF-PCR-Product, GC-PCR-product and SM-PCR-product

        :   with EcoRI and PstI

1% Agarose gel
:   [![](/wiki/images/9/9f/Syn_MR_Gel51.jpg)](/File%3ASyn_MR_Gel51.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel51.jpg "Enlarge")

    M:1kb gene ruler plus, 1-4:AmiC-Terminator, 5-6:GroES-Terminator, 7:Scar, 8: control, (colony-PCR 13.09.2012)

## 14.09.2012

Results
:   Transformation

    :   AmiC-T: no colonies
    :   GroES-T: no colonies

Restriction
:   2nd Digestion of A-modules (see digest from yesterday) additionally with EcoRI

Ligation
:   Gin-RBS with pSB1C3

    :   3 reactions:

        :   one with RBS-Gin HF-PCR-Product
        :   one with RBS-Gin GC-PCR-Product
        :   one with RBS-Gin SM-PCR-Product

Inoculation
:   Gix-K3 (clone 3 and 4)
:   P-RBS-C3 (clone 1 and 3)
:   RBS (BBa\_B0030) [clone 1 and 3]

1% Agarose gel
:   [![](/wiki/images/2/26/Syn_MR_Gel52.jpg)](/File%3ASyn_MR_Gel52.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel52.jpg "Enlarge")

    M:1kb gene ruler plus, 1:AmiC, 2:GroES, 3:none, 4:GFP, 5:CFP, 6:mRFP,(digest 13.-14.09.2012)

Gel extraction
:   GFP
:   mRFP

1% Agarose gel
:   [![](/wiki/images/3/3d/Syn_MR_Gel53.jpg)](/File%3ASyn_MR_Gel53.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel53.jpg "Enlarge")

    M:1kb gene ruler plus, 1:Terminator, (digest 14.09.2012)

Gel extraction
:   Terminator clone 48

Transformation
:   RBS-Gin(HF)-pSB1C3
:   RBS-Gin(GC)-pSB1C3
:   RBS-Gin(SM)-pSB1C3

# Week 11

17.09. - 23.09.2012

## 17.09.2012

Restriction
:   AmiC
:   GroES
:   CFP
:   SacB

1% Agarose gel
:   [![](/wiki/images/1/17/Syn_MR_Gel54.jpg)](/File%3ASyn_MR_Gel54.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel54.jpg "Enlarge")

    M:1kb gene ruler plus, 1:AmiC control, 2:AmiC, 3:GroES control, 4:GroES, 5:CFP control, 6:CFP, 7:SacB control, 8:SacB (digest 17.09.2012)

Restriction
:   CFP-C3
:   SacB (2nd try)

Resuspend
:   BBa\_B001J

Restreak
:   RBS-Gin (HF)
:   RBS-Gin (GC)
:   RBS-Gin (SM)

DNA isolation
:   CFP-C3 (Miniprep28.08.12)--> 47,3 ng/µl

Miniprep
:   GixR pSB1K3 --> 50,5 ng/µl, 144,7 ng/µl
:   RBS(new)B0030 pSB1A2 --> 726,2 ng/µl, 242,6 ng/µl
:   P108-RBS pSB1C3 --> 200,5 ng/µl, 177,8 ng/µl

1% Agarose gel
:   [![](/wiki/images/0/07/Syn_MR_Gel55.jpg)](/File%3ASyn_MR_Gel55.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel55.jpg "Enlarge")

    M:1kb gene ruler plus, 1-2:GixR, 3-4:RBS BBa\_B0030, 3-4:P108-RBS, (mini prep 17.09.2012)

Transformation
:   pSB2K3-pBAD & pSB1-A2-TT

1% Agarose gel
:   CFP-C3 (restriction 2nd try)
:   SacB-Cs (restriction 2nd try)
:   [![](/wiki/images/6/64/Syn_MR_Gel56.jpg)](/File%3ASyn_MR_Gel56.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel56.jpg "Enlarge")

    M:1kb gene ruler plus, 1:CFP control, 2:CFP, 3:SacB, (digest 17.09.2012)

## 18.09.2012

Results

Transformation
:   2x Terminator --> colonies
:   pBAD --> no colonies

Colony PCR
:   RBS-Gin
:   2x Terminator
:   parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")

1% Agarose gel
:   [![](/wiki/images/0/0a/Syn_MR_Gel57.jpg)](/File%3ASyn_MR_Gel57.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel57.jpg "Enlarge")

    M:1kb gene ruler plus, 1-2:RBS-Gin HF, 3:RBS-Gin GC, 4:RBS-Gin SM, 5: Scar, 7-10:2xTerminator, 11:control, (colony-pcr 18.09.2012)

Sequencing
:   SacB

Gel extraction
:   CFP-C3 E+X

Ligation
:   am-G: amiC E+S & GFP-A3 E+X
:   am-C: amiC E+S & CFP-C3 E+X
:   am-R: amiC E+S & mRFP-C3 E+X
:   gro-G: groES E+S & GFP-A3 E+X
:   gro-C: groES E+S & CFP-C3 E+X
:   gro-R: groES E+S & mRFP-C3 E+X

Transformation
:   am-G
:   am-C
:   am-R
:   gr-G
:   gr-C
:   gr-R
:   pBAD

## 19.09.2012

Results

Transformation
:   am-G --> colonies
:   am-C --> colonies
:   am-R --> colonies
:   gr-G --> colonies
:   gr-C --> colonies
:   gr-R --> colonies
:   pBAD --> no colonies

Colony PCR
:   RBS-Gin (from 17.09.12)
:   2x Terminator B0017 (from 17.09.12)
:   parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
:   primer [MS20](/Team%3AMarburg_SYNMIKRO/Primer#MS20 "Team:Marburg SYNMIKRO/Primer") + [MS21](/Team%3AMarburg_SYNMIKRO/Primer#MS21 "Team:Marburg SYNMIKRO/Primer")
:   --> Amplification failed

Transformation
:   pBAD

Microscopy-screening
:   AmiC-GFP --> fluorescence
:   GroES-GFP --> fluorescence
:   AmiC-CFP --> fluorescence
:   GroES-CFP --> fluorescence
:   AmiC-mRFP --> fluorescence
:   GroES-mRFP --> fluorescence

Inoculation
:   RBS-Gin
:   TT (B0017)
:   AmiC-GFP
:   GroES-GFP
:   AmiC-CFP
:   GroES-CFP
:   AmiC-mRFP
:   GroES-mRFP

## 20.09.2012

Miniprep
:   RBS-Gin
:   TT (B0017)
:   AmiC-GFP
:   GroES-GFP
:   AmiC-CFP
:   GroES-CFP
:   AmiC-mRFP
:   GroES-mRFP

Restriction (E/P)
:   TT(B0017)
:   RBS-Gin

1% Agarose gel
:   [![](/wiki/images/e/ee/Syn_MR_Gel58.jpg)](/File%3ASyn_MR_Gel58.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel58.jpg "Enlarge")

    M:1kb gene ruler plus, 1,3,5,7:2xTerminator, 2,4,6,8:2xTerminator control, 9,11,13,15:RBS-Gin, 10,12,14,16:RBS-Gin control, (control digest 20.09.2012)

Restriction
:   SacB ((XbaI + PstI) & (EcoRI + XbaI)
:   P-RBS (EcoRI + SpeI) & (SpeI + PstI)

1% Agarose gel
:   [![](/wiki/images/7/7f/Syn_MR_Gel59.jpg)](/File%3ASyn_MR_Gel59.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel59.jpg "Enlarge")

    M:1kb gene ruler plus, 1:P-RBS control, 2: P-RBS (E/S), 3: P-RBS (S/P), 4: SacB control, 5: SacB (X/P), 6:SacB (E/X), (digest 20.09.2012)

Inoculation
:   GroES-GFP
:   GroES-CFP
:   GroES-mRFP

## 21.09.2012

Restriction
:   GFP-fusion (EcoRI + SpeI)
:   TT BBa\_0017 (EcoRI + XbaI)

1% Agarose gel
:   [![](/wiki/images/a/ab/Syn_MR_Gel60.jpg)](/File%3ASyn_MR_Gel60.jpg)

    [![](/wiki/skins/common/images/magnify-clip.png)](/File%3ASyn_MR_Gel60.jpg "Enlarge")

    M:1kb gene ruler plus, 1:GFP-Fusion, 2:TT BBa\_B0017, 3:SacB, (digest 21.09.2012)

Gel extraction
:   GFP-Fusion
:   pSB1A2 (backbone GFP-Fusion)
:   TT
:   SacB

Ligation
:   GFP bb Enhancer
:   GFP TT
:   GroES TT

Transformation
:   GFP bb Enhancer
:   GFP TT
:   GroES TT
:   Promotor-RBS (17.09.12)

Sequencing
:   RBS-Gin

Microscopy-screening
:   AmiC-GFP --> Channel: DIC, Green, Red, Blue
:   GroES-GFP --> Channel: DIC, Green, Red, Blue
:   AmiC-CFP --> Channel: DIC, Green, Red, Blue
:   GroES-CFP --> Channel: DIC, Green, Red, Blue
:   AmiC-mRFP --> Channel: DIC, Red
:   GroES-mRFP --> Channel: DIC, Green, Red, Blue

## 22.09.2012

Inoculation
:   GroES-TT pSB1A2
:   GFP-TT pSB1A2
:   Enhancer
:   P108-RBS pSB1C3

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