Team:Marburg SYNMIKRO/Notebook

From 2012.igem.org

(Difference between revisions)
(Prototype team page)
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<!-- *** What falls between these lines is the Alert Box!  You can remove it from your pages once you have read and understood the alert *** -->
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{{:Team:Marburg_SYNMIKRO:Template:Header}}
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<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 3px solid #000; background-color: #fe2b33;">
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<style>
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<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: #f6f6f6; padding: 5px;">
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This is a template page. READ THESE INSTRUCTIONS.
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#toc {
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display:none;
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<div onClick="javascript:toggledisplay('klapp1'); return false">
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= Week 1: 09.07. - 15.07.2012 =
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<html>
</div>
</div>
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<div id="instructions" style="text-align: center; font-weight: normal; font-size: small; color: #f6f6f6; padding: 5px;">
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<div class="hiddenifjs" id="klapp1">
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You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples <a href="https://2008.igem.org/Help:Template/Examples">HERE</a>.
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</html>
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 +
 
 +
==09.07.2012==
 +
; Inoculation
 +
: ''E. coli'' strain C600 Mucts 62' in DYT-medium
 +
 
 +
==10.07.2012==
 +
; Isolation of chromosomal DNA
 +
: ''E. coli'' strain C600 Mucts 62'''
 +
; Inoculation
 +
: ''E. coli'' strain DH5a 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
 +
 
 +
{|class="wikitable"  
 +
!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
 +
|}
 +
 
 +
 
 +
 
 +
<html>
</div>
</div>
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<div id="warning" style="text-align: center; font-weight: bold; font-size: small; color: #f6f6f6; padding: 5px;">
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</html>
-
You <strong>MUST</strong> have all of the pages listed in the menu below with the names specified. PLEASE keep all of your pages within your teams namespace.
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 +
<html>
 +
<div onClick="javascript:toggledisplay('klapp2'); return false">
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</html>
 +
= Week 2: 16.07. - 22.07.2012 =
 +
<html>
</div>
</div>
 +
<div  class="hiddenifjs" id="klapp2">
 +
</html>
 +
 +
==16.07.2012==
 +
; PCR
 +
: AmiC (primer: MS1 + MS2)
 +
: HU (primer: MS3 + MS4)
 +
: GroES (primer: MS5 + MS6)
 +
: GixR (primer: MS7 + MS8)
 +
: Enhancer (primer: MS9 + MS10)
 +
: &rArr; template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 30 sec; 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
 +
:<strike>LacIQ</strike>
 +
:P100
 +
 +
==18.07.2012==
 +
; Agarose gel
 +
: pipet scheme
 +
Marker|enhancer|GroES|AmiC|HU|GixR|GFP|GFP-Fusion|CFP|mRFP|RBS|LacIQ|J23108|J23100|λ-DNA
 +
(bild 1)
 +
 +
; DNA Gel extraction
 +
: Enhancer
 +
: GroES
 +
: HU
 +
 +
; GixR Hybridization
 +
* 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (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 + MS2)
 +
 +
 +
: &rArr; 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
 +
: 7) CFP (EcoRI/PstI)
 +
: 8) GFP (EcoRI/PstI)
 +
: 9) GFP-fusion (EcoRI/PstI)
 +
: 10) mRFP (EcoRI/PstI)
 +
: &rArr; all negative
 +
 +
; Agarose gel
 +
: pipet scheme
 +
1)|2)|3)|4)|5)|marker|7)|8)|9)|10)
 +
(bild 2)
 +
; Control digest
 +
: CFP (EcoRI/PstI)
 +
: GFP (EcoRI/PstI)
 +
: GFP-fusion (EcoRI/PstI)
 +
: mRFP (EcoRI/PstI)
 +
: &rArr; restriction over night
 +
 +
==20.07.2012==
 +
; Agarose gel
 +
: pipet scheme
 +
: &rArr; marker|GFP|GFP-fusion|CFP|mRFP (all positive)
 +
(bild 3)
 +
 +
 +
<html>
</div>
</div>
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</html>
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<!-- *** End of the alert box *** -->
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<html>
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<div onClick="javascript:toggledisplay('klapp3'); return false">
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</html>
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= Week 3: 23.07. - 29.07.2012 =
 +
<html>
 +
</div>
 +
<div  class="hiddenifjs" id="klapp3">
 +
</html>
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
+
==26.07.2012==
-
!align="center"|[[Team:Marburg_SYNMIKRO|Home]]
+
; Restriction
-
!align="center"|[[Team:Marburg_SYNMIKRO/Team|Team]]
+
: pSB1T3 (XbaI/SpeI and DpnI)
-
!align="center"|[https://igem.org/Team.cgi?year=2012&team_name=Marburg_SYNMIKRO Official Team Profile]
+
: pSB1A3 (XbaI/SpeI and DpnI)
-
!align="center"|[[Team:Marburg_SYNMIKRO/Project|Project]]
+
: pSB1K3 (XbaI/SpeI and DpnI)
-
!align="center"|[[Team:Marburg_SYNMIKRO/Parts|Parts Submitted to the Registry]]
+
: pSB1C3 (XbaI/SpeI and DpnI)
-
!align="center"|[[Team:Marburg_SYNMIKRO/Modeling|Modeling]]
+
-
!align="center"|[[Team:Marburg_SYNMIKRO/Notebook|Notebook]]
+
-
!align="center"|[[Team:Marburg_SYNMIKRO/Safety|Safety]]
+
-
!align="center"|[[Team:Marburg_SYNMIKRO/Attributions|Attributions]]
+
-
|}
+
 +
; Ligation
 +
: GixR + pSB1A3
 +
: Enhancer + pSB1K3
 +
: cyclisation of pSB1C3, pSB1K3, pSB1A3, pSB1T3 (couldn’t work)
-
You should make use of the calendar feature on the wiki and start a lab notebook.  This may be looked at by the judges to see how your work progressed throughout the summer.  It is a very useful organizational tool as well.
+
 
 +
<html>
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</div>
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</html>
 +
 
 +
<html>
 +
<div onClick="javascript:toggledisplay('klapp4'); return false">
 +
</html>
 +
= Week 4: 30.07. - 05.08.2012 =
 +
<html>
 +
</div>
 +
<div  class="hiddenifjs" id="klapp4">
 +
</html>
 +
 
 +
 
 +
==30.07.2012==
 +
 
 +
; PCR
 +
: CFP (primer: MS16 + MS17) &rArr; template-DNA: BBa_E0010
 +
: &rArr; positive
 +
: mRFP (primer: MS14 + MS15) &rArr; template-DNA: BBa_E1010
 +
: &rArr; positive
 +
: Gin (primer: MS11 + MS13) &rArr; template-DNA: chromosomal DNA (that was wrong)
 +
: RBS-Gin (primer: MS13 + MS12) &rArr; template-DNA: chromosomal DNA (that was wrong)
 +
: &rArr; 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
 +
 
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: &rArr; gix|marker|AmiC from 16.07.| AmiC from 16.07.| AmiC from 19.07.| AmiC from 19.07.
 +
: &rArr; all negative
 +
(bild 4,5)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: &rArr; marker|RBS-Gin from 30.07.|AmiC from 16.07.|Gin from 30.07.|CFP from 30.07.|mRFP from 30.07.|control
 +
: &rArr; all negative
 +
 
 +
; Resuspension of a biobrick
 +
: BBa_B0010 (terminator)
 +
 
 +
; DNA Gel extraction
 +
: CFP
 +
: mRFP
 +
 
 +
; PCR
 +
: AmiC (primer: MS1 + MS2)
 +
: RBS-Gin (primer: MS12 + MS13)
 +
: Gin (primer: MS11 + MS13)
 +
: &rArr; 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 (100 µM) + 5 µl primer MS8 (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 &rArr; only clone 1, 3 Positive
 +
: pSB1C3 &rArr; all negative
 +
: pSB1K3 &rArr; all negative
 +
 
 +
; Ligation
 +
: GixR hyb. + pJET2_1
 +
: &rArr; over blunt ends
 +
 
 +
; Restriction
 +
: GixR hyb. (EcoRI/PstI)
 +
: pSB1C3 (EcoRI/PstI)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|RBS-Gin1 +DMSO|RBS-Gin2 -DMSO|Gin1 +DMSO|Gin2 -DMSO|control
 +
(bild 6)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|AmiC1 +DMSO| AmiC2 –DMSO
 +
(bild 7)
 +
 
 +
; 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
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker| pSB1A3 1,2,3| pSB1C3 1,2,3,4| pSB1K3 1,2,3,4
 +
(bild 8)
 +
: 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
 +
: &rArr; for ccdB
 +
 
 +
; Plasmid preparation
 +
: BBa_B0010 (terminator)
 +
 
 +
; Test restriction
 +
: Terminator c1,2,3,4 (EcoRI/PstI)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|Terminator 1,2,3,4
 +
(bild 9)
 +
 
 +
; Transformation
 +
: pSB1T3 recycled
 +
: AmiC in T3
 +
: Gin-RBS in K3
 +
: Gin in K3
 +
: pSB3C5 linear (false)
 +
 
 +
 
 +
==03.08.2012==
 +
 
 +
; Plasmid preparation
 +
: Enhancer in pJET
 +
 
 +
 
 +
 
 +
 
 +
<html>
 +
</div>
 +
</html>
 +
 
 +
<html>
 +
<div onClick="javascript:toggledisplay('klapp5'); return false">
 +
</html>
 +
= Week 5: 06.08. - 12.08.2012 =
 +
<html>
 +
</div>
 +
<div class="hiddenifjs" id="klapp5">
 +
</html>
 +
 
 +
 
 +
==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 (100 µM) + 1(3) µl primer MS8 (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)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|pSB3C5 c1a,2a,3a,4a|Marker|pSB3C5 c1b,2b,3b,4b|Marker|Enhancer-pJET c1,2,3,4|Marker
 +
(bild 10)
 +
: &rArr; positive: pSB3C5 c3a,4a,3b,4b,; Enhancer-pJET c1,2,3,4
 +
 
 +
==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)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker| Enhancer|pSB1C3
 +
(bild 11)
 +
 
 +
; 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 &rArr; positive
 +
: J23108-RBS &rArr; positive
 +
: J23100-RBS &rArr; positive
 +
: CFP &rArr; positive
 +
: GixR in C3 c1 &rArr; negative
 +
: GixR in C3 c2 &rArr; negative
 +
: GixR in C3 c3 &rArr; 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)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|GFP-fusion 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
 +
(bild 12)
 +
 
 +
: Marker|J23108-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
 +
(bild 13)
 +
 
 +
: Marker|J23100-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18
 +
(bild 14)
 +
 
 +
: Marker|GFP-fusion 19,20| J23108-RBS 19,20| J23100-RBS 19,20| Marker |CFP 1,2,3,4,5
 +
(bild 15)
 +
 
 +
 
 +
; Inoculation
 +
: GixR in C3
 +
: Gin in T3
 +
: Gin-RBS in T3
 +
: pSB1C3
 +
: Enhancer in C3
 +
: mRFP in A3
 +
 
 +
 
 +
<html>
 +
</div>
 +
</html>
 +
 
 +
<html>
 +
<div onClick="javascript:toggledisplay('klapp6'); return false">
 +
</html>
 +
= Week 6: 13.08.2012 - 19.08.2012 =
 +
<html>
 +
</div>
 +
<div class="hiddenifjs" id="klapp6">
 +
</html>
 +
 
 +
 
 +
== 13.08.12 ==
 +
 
 +
; PCR
 +
: GroES (primer: MS5 + MS6 (b))
 +
: HU (primer: MS3 + MS4 (b))
 +
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO|control
 +
(bild 16)
 +
 
 +
; 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 + MS6 (b))
 +
: HU (primer: MS3 + MS4 (b))
 +
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C or annealing/elongation: 30 sec;  72 °C, 35 cycles
 +
 
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO| HU +DMSO two-step PCR|HU -DMSO two-step PCR |GroES +DMSO two-step PCR |GroES –DMSO two-step PCR
 +
(bild 17)
 +
 
 +
 
 +
; Resuspension of a biobrick
 +
: BBa_J04450 in pSB1A3
 +
: BBa_J04450 in pSB1T3
 +
: BBa_J04450 in pSB1C3
 +
: BBa_J04450 in pSB1K3
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker|HU -DMSO|HU +DMSO|GroES -DMSO|GroES +DMSO
 +
(bild 18)
 +
 
 +
: &rArr; 2-step PCR didn’t work, HU 1,2
 +
 
 +
; DNA Gel extraction
 +
: HU 1,2
 +
 
 +
; 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 + MS6 (b))
 +
: &rArr; with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: Marker| GroES 1, 2 +DMSO
 +
(bild 19)
 +
 
 +
; Plasmid preparation
 +
: P-RBS
 +
 
 +
; Test restriction
 +
: P-RBS (EcoRI/PstI)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: control|P-RBS|marker
 +
(bild 20)
 +
 
 +
 
 +
; 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)
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: marker|BBa_J04450 in pSB1A3 1,2,3,4|BBa_J04450 in pSB1K3 1,2,3,4| marker| BBa_J04450 in pSB1C3 1,2,3,4
 +
(bild 21)
 +
 
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: marker|P-RBS 1,2,3,4| P-RBS 1,2,3,4| marker| BBa_J04450 in pSB1K3 1,2,3,4
 +
 
 +
(bild 22)
 +
 
 +
 
 +
<html>
 +
</div>
 +
</html>
 +
 
 +
<html>
 +
<div onClick="javascript:toggledisplay('aufklapp6'); return false">
 +
</html>
 +
= Week 7: 20.08.2012 - 26.08.2012 =
 +
<html>
 +
</div>
 +
<div id="aufklapp6">
 +
</html>
 +
 
 +
 
 +
== 20.08.2012 ==
 +
 
 +
; PCR
 +
: Backbone (primer MS24 + MS25)
 +
: &rArr; template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
 +
 
 +
; Agarose gel
 +
: pipet scheme
 +
: marker|: pSB1A3|pSB1T3|pSB1C3|pSB1K3
 +
(bild 23)
 +
 
 +
; 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 --> MS24Prefix_rev + MS25Suffix_fwd
 +
: 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 - ∞
 +
 
 +
; miniprep
 +
: GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old)
 +
 
 +
; Test restriction
 +
: of miniprep
 +
: over night, 37°C
 +
 
 +
; Agarose gel (1%)
 +
: watch pic
 +
 
 +
; Inoculation
 +
: E.coli BBa_I14032
 +
: over night, 37°C
 +
 
 +
 
 +
== 22.08.2012 ==
 +
 
 +
; PCR
 +
: CFP
 +
: mRFP
 +
: 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 - ∞
 +
 
 +
; Agarose gel (1%)
 +
: watch picture
 +
 
 +
; miniprep
 +
: of LacIQ
 +
 
 +
; Test restriction
 +
: of LacIQ with EcoRI and PstI
 +
: watch picture
 +
 
 +
; Agarose gel
 +
: test restriction of GroES and GFP-T (->21.08.12), gel 1%
 +
: watch picture
 +
: 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 ==
 +
 
 +
Svetti please check!
 +
-->lacIQ evtl ganz rausnehmen?! ;)
 +
 
 +
== 24.08.2012 ==
 +
 
 +
Svetti please check!
 +
 
 +
 
 +
<html>
 +
</div>
 +
</html>
 +
 
 +
<html>
 +
<div onClick="javascript:toggledisplay('aufklapp7'); return false">
 +
</html>
 +
 
 +
= Week 8: 27.08.2012 - 02.09.2012 =
 +
<html>
 +
</div>
 +
<div id="aufklapp7">
 +
</html>
 +
 
 +
 
 +
== 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 ∞
 +
 
 +
; colony-PCR
 +
: parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
 +
 
 +
; Agarose gel of backbone PCR
 +
: no result
 +
 
 +
; elution of the backbones of Friday the 24th
 +
 
 +
; Agarose gel of colony PCR
 +
: watch picture
 +
 
 +
; 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 ~
 +
: watch picture
 +
 
 +
; Transformation
 +
: pBad, araC, 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
 +
: watch picture
 +
 
 +
; 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
 +
: bitte mal nachschlagen, die Notizen peil ich nicht ;)
 +
 
 +
 
 +
== 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
 +
 
 +
; Agarose gel
 +
: watch picture
 +
: cut out lines at 1.5kb
 +
 
 +
; 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
 +
 
 +
 
 +
<html>
 +
</div>
 +
</html>
 +
 
 +
<html>
 +
<div onClick="javascript:toggledisplay('aufklapp8'); return false">
 +
</html>
 +
= Week 9: 03.09.2012 - 09.09.2012 =
 +
<html>
 +
</div>
 +
<div id="aufklapp8">
 +
</html>
 +
 
 +
 
 +
== 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
 +
 
 +
 
 +
; PCR
 +
: of colonies (CFP-T, mRFP-T)
 +
: parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
 +
 
 +
; 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)
 +
: result watch picture
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<html>
 +
</div>
 +
</html>
 +
 
 +
<html>
 +
<div onClick="javascript:toggledisplay('aufklapp9'); return false">
 +
</html>
 +
= Week 10: 10.09.2012 - 16.09.2012 =
 +
<html>
 +
</div>
 +
<div id="aufklapp9">
 +
</html>
 +
 
 +
== 11.09.2012 ==
 +
;microscopy-screening
 +
:GroES-GFP &rArr;; no fluorescence
 +
:GroES-CFP &rArr;; no fluorescence
 +
:AmiC-mRFP &rArr;; no fluorescence
 +
:AmiC-GFP &rArr;; no fluorescence
 +
 
 +
;colony-PCR
 +
:GixR
 +
:RBS-Gin
 +
:Terminator
 +
 
 +
;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
 +
 
 +
<html>
 +
</div>
 +
</script>
 +
<script type="text/javascript">
 +
 
 +
function ReplaceContentInContainer(matchClass)
 +
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            elems[i].style.display = 'none';
 +
            }
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 +
window.onload = function ()
 +
    {
 +
    ReplaceContentInContainer("hiddenifjs");
 +
    }
 +
</script>
 +
</html>
 +
 
 +
{{:Team:Marburg_SYNMIKRO:Template:Footer}}

Revision as of 10:57, 24 September 2012

Main Page

Contents

Week 1: 09.07. - 15.07.2012


09.07.2012

Inoculation
E. coli strain C600 Mucts 62' in DYT-medium

10.07.2012

Isolation of chromosomal DNA
E. coli strain C600 Mucts 62
Inoculation
E. coli strain DH5a 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
NameBiobrickPlateWellBackboneResistance
GFP[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]114KpSB1A2Ampicillin
GFP_fusion[http://partsregistry.org/Part:BBa_K125500 BBa_K125500]32PpSB1A2Ampicillin
CFP[http://partsregistry.org/Part:BBa_E0020 BBa_E0020]16ApSB1A2Ampicillin
mRFP[http://partsregistry.org/Part:BBa_E1010 BBa_E1010]118FpSB2K3Kanamycin
RBS[http://partsregistry.org/Part:BBa_J61100 BBa_J61100]15JpSB1A2Ampicillin
P108[http://partsregistry.org/Part:BBa_J23108 BBa_J23108]22EBBa_J61002Ampicillin
P100[http://partsregistry.org/Part:BBa_J23100 BBa_J23100]118CBBa_J61002Ampicillin
PLac[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]211PpSB2K3Kanamycin


Week 2: 16.07. - 22.07.2012

16.07.2012

PCR
AmiC (primer: MS1 + MS2)
HU (primer: MS3 + MS4)
GroES (primer: MS5 + MS6)
GixR (primer: MS7 + MS8)
Enhancer (primer: MS9 + MS10)
⇒ template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 30 sec; 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

Agarose gel
pipet scheme

Marker|enhancer|GroES|AmiC|HU|GixR|GFP|GFP-Fusion|CFP|mRFP|RBS|LacIQ|J23108|J23100|λ-DNA (bild 1)

DNA Gel extraction
Enhancer
GroES
HU
GixR Hybridization
  • 5 µl primer MS7 (100 µM) + 5 µl primer MS8 (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 + MS2)


⇒ 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
7) CFP (EcoRI/PstI)
8) GFP (EcoRI/PstI)
9) GFP-fusion (EcoRI/PstI)
10) mRFP (EcoRI/PstI)
⇒ all negative
Agarose gel
pipet scheme

1)|2)|3)|4)|5)|marker|7)|8)|9)|10) (bild 2)

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

20.07.2012

Agarose gel
pipet scheme
⇒ marker|GFP|GFP-fusion|CFP|mRFP (all positive)

(bild 3)


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)


Week 4: 30.07. - 05.08.2012


30.07.2012

PCR
CFP (primer: MS16 + MS17) ⇒ template-DNA: BBa_E0010
⇒ positive
mRFP (primer: MS14 + MS15) ⇒ template-DNA: BBa_E1010
⇒ positive
Gin (primer: MS11 + MS13) ⇒ template-DNA: chromosomal DNA (that was wrong)
RBS-Gin (primer: MS13 + MS12) ⇒ 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


Agarose gel
pipet scheme
⇒ gix|marker|AmiC from 16.07.| AmiC from 16.07.| AmiC from 19.07.| AmiC from 19.07.
⇒ all negative

(bild 4,5)

Agarose gel
pipet scheme
⇒ marker|RBS-Gin from 30.07.|AmiC from 16.07.|Gin from 30.07.|CFP from 30.07.|mRFP from 30.07.|control
⇒ all negative
Resuspension of a biobrick
BBa_B0010 (terminator)
DNA Gel extraction
CFP
mRFP
PCR
AmiC (primer: MS1 + MS2)
RBS-Gin (primer: MS12 + MS13)
Gin (primer: MS11 + MS13)
⇒ 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 (100 µM) + 5 µl primer MS8 (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)
Agarose gel
pipet scheme
Marker|RBS-Gin1 +DMSO|RBS-Gin2 -DMSO|Gin1 +DMSO|Gin2 -DMSO|control

(bild 6)

Agarose gel
pipet scheme
Marker|AmiC1 +DMSO| AmiC2 –DMSO

(bild 7)

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
Agarose gel
pipet scheme
Marker| pSB1A3 1,2,3| pSB1C3 1,2,3,4| pSB1K3 1,2,3,4

(bild 8)

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)
Agarose gel
pipet scheme
Marker|Terminator 1,2,3,4

(bild 9)

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 (100 µM) + 1(3) µl primer MS8 (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)
Agarose gel
pipet scheme
Marker|pSB3C5 c1a,2a,3a,4a|Marker|pSB3C5 c1b,2b,3b,4b|Marker|Enhancer-pJET c1,2,3,4|Marker

(bild 10)

⇒ positive: pSB3C5 c3a,4a,3b,4b,; Enhancer-pJET c1,2,3,4

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)
Agarose gel
pipet scheme
Marker| Enhancer|pSB1C3

(bild 11)

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)
Agarose gel
pipet scheme
Marker|GFP-fusion 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18

(bild 12)

Marker|J23108-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18

(bild 13)

Marker|J23100-RBS 1,2,3,4,5,6,7,8,9| Marker|10,11,12,13,14,15,16,17,18

(bild 14)

Marker|GFP-fusion 19,20| J23108-RBS 19,20| J23100-RBS 19,20| Marker |CFP 1,2,3,4,5

(bild 15)


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


Week 6: 13.08.2012 - 19.08.2012


13.08.12

PCR
GroES (primer: MS5 + MS6 (b))
HU (primer: MS3 + MS4 (b))
⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 60 °C, elongation: 40 sec; 72 °C, 35 cycles
Agarose gel
pipet scheme
Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO|control

(bild 16)

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 + MS6 (b))
HU (primer: MS3 + MS4 (b))
⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C or annealing/elongation: 30 sec; 72 °C, 35 cycles


Agarose gel
pipet scheme
Marker|HU +DMSO|HU -DMSO|GroES +DMSO|GroES –DMSO| HU +DMSO two-step PCR|HU -DMSO two-step PCR |GroES +DMSO two-step PCR |GroES –DMSO two-step PCR

(bild 17)


Resuspension of a biobrick
BBa_J04450 in pSB1A3
BBa_J04450 in pSB1T3
BBa_J04450 in pSB1C3
BBa_J04450 in pSB1K3
Agarose gel
pipet scheme
Marker|HU -DMSO|HU +DMSO|GroES -DMSO|GroES +DMSO

(bild 18)

⇒ 2-step PCR didn’t work, HU 1,2
DNA Gel extraction
HU 1,2
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 + MS6 (b))
⇒ with and without DMSO, template-DNA: chromosomal DNA, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
Agarose gel
pipet scheme
Marker| GroES 1, 2 +DMSO

(bild 19)

Plasmid preparation
P-RBS
Test restriction
P-RBS (EcoRI/PstI)
Agarose gel
pipet scheme
control|P-RBS|marker

(bild 20)


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)
Agarose gel
pipet scheme
marker|BBa_J04450 in pSB1A3 1,2,3,4|BBa_J04450 in pSB1K3 1,2,3,4| marker| BBa_J04450 in pSB1C3 1,2,3,4

(bild 21)


Agarose gel
pipet scheme
marker|P-RBS 1,2,3,4| P-RBS 1,2,3,4| marker| BBa_J04450 in pSB1K3 1,2,3,4

(bild 22)


Week 7: 20.08.2012 - 26.08.2012


20.08.2012

PCR
Backbone (primer MS24 + MS25)
⇒ template-DNA: pSB1A3, pSB1T3, pSB1C3 and pSB1K3, annealing: 30 sec; 55 °C, elongation: 30 sec; 72 °C , 35 cycles
Agarose gel
pipet scheme
marker|: pSB1A3|pSB1T3|pSB1C3|pSB1K3

(bild 23)

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 --> MS24Prefix_rev + MS25Suffix_fwd
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 - ∞
miniprep
GroES, Enh 3/4 (+old), P-RBS (+old), GFP-T (+old)
Test restriction
of miniprep
over night, 37°C
Agarose gel (1%)
watch pic
Inoculation
E.coli BBa_I14032
over night, 37°C


22.08.2012

PCR
CFP
mRFP
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 - ∞
Agarose gel (1%)
watch picture
miniprep
of LacIQ
Test restriction
of LacIQ with EcoRI and PstI
watch picture
Agarose gel
test restriction of GroES and GFP-T (->21.08.12), gel 1%
watch picture
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

Svetti please check! -->lacIQ evtl ganz rausnehmen?! ;)

24.08.2012

Svetti please check!


Week 8: 27.08.2012 - 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 ∞
colony-PCR
parameters: 98°C 5min, >> 98°C 1min, 72°C 1min << x30, 72°C 5min, 4°C ∞
Agarose gel of backbone PCR
no result
elution of the backbones of Friday the 24th
Agarose gel of colony PCR
watch picture
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 ~
watch picture
Transformation
pBad, araC, 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
watch picture
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
bitte mal nachschlagen, die Notizen peil ich nicht ;)


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
Agarose gel
watch picture
cut out lines at 1.5kb
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: 03.09.2012 - 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


PCR
of colonies (CFP-T, mRFP-T)
parameters: 98°C 5', >>98°C 1', 72°C 1'<<x30, 72°C 5', 4°C
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)
result watch picture




Week 10: 10.09.2012 - 16.09.2012

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
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

Retrieved from "http://2012.igem.org/Team:Marburg_SYNMIKRO/Notebook"