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September

rMFC

CO₂ fixation

Isobutanol

Biosafety

General

Week 1 09/01 - 09/07

adhA

This week we tried to verify the positive cutures that were identified last week by a restriction digest

Plasmid isolation of pSB1K3_adhA

Restriction digestion with EcoRI and PstI of pSB1K3_adhA

Bands as expected (~1200 bp)

Agarose gel from the restriction digestion of pSB1K3_adhA with EcoRI and PstI. As a Ladder we used the GeneRuler™ 1 kb DNA Ladder. The fragments in both lines have the right sizes..

Successful sequencing

T7_alsS_ilvC_ilvD_kivD

This week we tried to verify the positive cutures that were identified last week by a restriction digest

Plasmid isolation of pSB1A2_T7_alsS_ilvC_ilvD_kivD

Restriction digestion with EcoRI and PstI of pSB1A2_T7_alsS_ilvC_ilvD_kivD

Bands not as expected (Backbone: ~ 2200 and insert: ~ 7500bp)

pSB1K3_alsS_ilvC_ilcD_kivD_adhA

This week we tried to combine the pSB1K3_alsS_ilvC_ilvD_kivD with adhA

BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

pSB1K3_alsS_ilvC_ilvD_kivD

Insert (digested with XbaI, PstI)

pSB1K3_alsS_ilvC_ilvD_kivD_adhA

Colony PCR (fw-ilvD-kivD, rev-pSB1C3-adhA)

Annealing temperature: 65 °C
Bands not as expected (~ 3800 bp)

Week 2 09/08 - 09/14

pSB1C3_alsS_ilvC_ilvD_kivD

This week we tried to reclone alsS_ilvC_ilvD_kivD into pSB1C3

Restriction digestion of pSB1K3_alsS_ilvC_ilvD_kivD and pSB1C3_RFP with EcoRI and PstI

Bands as expected
(~ 7500 bp for alsS_ilvC_ilvD_kivD and ~ 2200 bp for pSB1C3)

Clean up and Ligation as explained in the BioBrick Assembly
Transformation with electrocompotetent cells

Colony PCR (fw_ilvD_kivD, rv_pSB1C3_kivD)

Annealing temperature: 65 °C
Bands not as expected (~ 1800 bp)

pSB1C3_alsS_ilvC_ilvD_kivD_adhA

This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly

NEB BioBrick Assembly

Upstream part pSB1K3_alsS_ilvC_ilvD_kivD (digested with EcoRI, SpeI)

alsS_ilvC_ilvD_kivD

Downstream part pSB1K3_adhA(digested with XbaI, PstI)

adhA

Destination part pSB1C3_RFP (digested with EcoRI, PstI)

alsS_ilvC_ilvD_kivD

Colony PCR (fw_ilvD_kivD , rev_pSB1C3_adhA)

Annealing temperature: 65°C
Bands not as expected (~ 3000 bp)

Week 3 09/15 - 09/21

pSB1C3_alsS_ilvC_ilvD_kivD

This week we tried to reclone alsS_ilvC_ilvD_kivD into pSB1C3

Restriction digestion of pSB1K3_alsS_ilvC_ilvD_kivD and pSB1C3_RFP with EcoRI and PstI

Bands as expected
(~ 7500 bp for alsS_ilvC_ilvD_kivD and ~ 2200 bp for pSB1C3)

Clean up and Ligation as explained in the BioBrick Assembly
Transformation with electrocompotetent cells

Colony PCR (fw_ilvD_kivD, rv_pSB1C3_kivD)

Annealing temperature: 65 °C
Bands not as expected (~ 1800 bp)

pSB1C3_adhA

This week we tried to reclone adhA into pSB1C3

Restriction digestion of pSB1K3_adhA and pSB1C3_RFP with EcoRI and PstI

Bands as expected
(~ 1200 bp for adhA and ~ 2200 bp for pSB1C3)

Clean up and Ligation as explained in the BioBrick Assembly
Transformation with electrocompotetent cells

Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)

Annealing temperature: 65 °C
Bands not as expected (~ 1200 bp)

pSB1A2_T7_adhA

This week we tried to contruct the pSB1A2_T7_adhA-Plasmid

BioBrick Assembly (Suffix)

Backbone pSB1A2_T7(digested with SpeI, PstI)

pSB1A2_T7

Insert pSB1K3_adhA(digested with XbaI, PstI)

adhA

Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)

Annealing temperature: 65 °C
Bands not as expected (~ 1200 bp)

pSB1C3_alsS_ilvC_ilvD_kivD_adhA

This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly

NEB BioBrick Assembly

Upstream part pSB1K3_alsS_ilvC_ilvD_kivD (digested with EcoRI, SpeI)

alsS_ilvC_ilvD_kivD

Downstream part pSB1K3_adhA(digested with XbaI, PstI)

adhA

Destination part pSB1C3_RFP (digested with EcoRI, PstI)

alsS_ilvC_ilvD_kivD

Colony PCR (fw_ilvD_kivD , rev_pSB1C3_adhA)

Annealing temperature: 65°C
Bands not as expected (~ 3000 bp)

Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the PCR purification. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. Thereby we discovered that the pSB1K3_alsS_ilvC_ilvD_kivD has an illigal restriction side.
Because of the integration of RBS's between all genes in the alsS_ilvC_ilvD_kivD-Plasmid, a restriction side between alsS and a RBS occurs.
New primers were ordered: rv_ilvC_alsS-new and fw_alsS_ilvC-new

Week 4 09/22 - 09/28

pSB1C3_alsS_ilvC_ilvD_kivD

This week the new primers rv_ilvC_alsS-new and fw_alsS_ilvC-new arrived. We tried to remove the illegale restriction side.

We tried different PCR amplifications to increase the chance to get a correct BioBrick. Therefore we amplified five parts for two combination possibilities in the Gibson Assembly:
1. fw_kivD_pSB1C3, rv_alsS_pSB1C3 on pSB1C3_RFP
  → pSB1C3 with ~ 2200 bp
2. fw_pSB1C3_alsS, rv_ilvC_alsS-new on pSB1K3_alsS_ilvC_ilvD_kivD
  → alsS with ~ 1800 bp
3. rv_pSB1C3_kivD, fw_alsS_ilvC-new on pSB1K3_alsS_ilvC_ilvD_kivD
  → ilvC_ilvD_kivD with ~ 5200 bp
4. rv_kivD_ilvD, fw_alsS_ilvC-new on pSB1K3_alsS_ilvC_ilvD_kivD
  → ilvC_ilvD with ~ 3600 bp
5. fw_ilvD_kivD, rv_pSB1C3_kivD on pSB1K3_alsS_ilvC_ilvD_kivD
  → kivD with ~ 1800 bp
- Annealing temperature: 65 °C
- Bands as expected (see above)

PCR products were purified out of the gel

Gibson Assembly with the previous PCR products:
- 1., 2. and 3.
- 1., 2., 4. and 5.

Transformation with electrocompotetent cells

Colony PCR (fw_pSB1C3_alsS, rv_ilvC_alsS-new)

Annealing temperature: 65 °C
Bands as expected (~ 1800 bp)

Liquid cultures for a restriction digest were prepared.
Plasmid isolation of pSB1C3-alsS-ilvC-ilvD-kivD without illigale restriction side
Restriction digestion with XbaI and PstI

Bands as expected (backbone: 2,2 kb and insert: 7 kb)

Successful sequencing

pSB1C3_adhA

This week we tried to construct the pSB1C3_adhA using Gibson Assembly.
PCR amplification of the pSB1C3_RFP for pSB1C3 as backbone (fw_adhA_pSB1C3, rev_adhA_pSB1C3) and pSB1K3_adhA for adhA as insert (rev_pSB1C3_adhA, fw_pSB1C3_adhA)

Annealing temperature: 65 °C
Bands as expected (~ 1200 bp for the adhA and ~ 2200 bp for the backbone)

PCR products were purified out of the gel
Gibson Assembly with adhA and pSB1C3
Transformation with electrocompotetent cells
Colony PCR (rev_pSB1C3_adhA, fw_pSB1C3_adhA)

Annealing temperature: 65 °C
Bands as expected (~ 1200 bp)

Liquid cultures for a restriction digest were prepared.
Plasmid isolation of pSB1C3-adhA
Restriction digestion with EcoRI and PstI

Bands as expected (backbone: 2,2 kb and insert: 1,2 kb)

Successful sequencing

Week 5 09/29 - 10/05

pSB1C3_ptac_alsS_ilvC_ilcD_kivD

This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the ptac promotor.
BioBrick Assembly (Suffix and Prefix)

Backbone (digested with SpeI, PstI)

pSB1C3_ptac

Insert (digested with XbaI, PstI)

alsS_ilvC_ilvD_kivD

Backbone (digested with EcoRI, XbaI)

alsS_ilvC_ilvD_kivD_pSB1C3

Insert (digested with EcoRI, SpeI)

ptac

Transformation with electrocompotetent cells
Colony PCR (VF-Primer, rv_ilvC_alsS-new)

Annealing temperature: 55°C
Bands as expected (~ 3000 bp)

Liquid culture for a restriction digest was prepared.
Plasmid isolation of pSB1C3-ptac_alsS_ilvC_ilvD_kivD
Restriction digestion with XbaI and PstI

Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)

Successful sequencing

pSB1C3_ptac_adhA

This week we tried to combine the pSB1C3_adhA contruct with the ptac promotor.
BioBrick Assembly (Suffix and Prefix)

Backbone (digested with SpeI, PstI)

pSB1C3_ptac

Insert (digested with XbaI, PstI)

adhA

Backbone (digested with EcoRI, XbaI)

adhA_pSB1C3

Insert (digested with EcoRI, SpeI)

ptac

Transformation with electrocompotetent cells
Colony PCR (VF-Primer, rev_pSB1C3_adhA)

Annealing temperature: 55°C
Bands as expected (~ 2800 bp)

Liquid cultures for a restriction digest were prepared.
Plasmid isolation of pSB1C3-adhA
Restriction digestion with XbaI and PstI

Bands as expected (backbone: ~ 2200 kb and insert: ~ 2400 bp)

Successful sequencing

pSB1C3_alsS_ilvC_ilcD_kivD_adhA

This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the adhA from L. lactis.
BioBrick Assembly (Suffix and Prefix)

Backbone (digested with SpeI, SpeI)

pSB1C3_adhA

Insert (digested with EcoRI, PstI)

alsS_ilvC_ilvD_kivD

Backbone (digested with SpeI, PstI)

pSB1C3_alsS_ilvC_ilvD_kivD

Insert (digested with XbaI, PstI)

ptac

Transformation with electrocompotetent cells
Colony PCR (fw_ilvD_kivD, rev_pSB1C3_adhA)

Annealing temperature: 55°C
Bands as expected (~ 3000 bp)

Liquid culture for a restriction digest was prepared.
Plasmid isolation of pSB1C3_alsS_ilvC_ilvD_kivD_adhA
Restriction digestion with EcoRI and PstI

Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)

Successful sequencing

pSB1A2_T7_adhA

This week we tried to contruct the pSB1A2_T7_adhA-Plasmid

BioBrick Assembly (Suffix)

Backbone pSB1A2_T7(digested with SpeI, PstI)

pSB1A2_T7

Insert pSB1C3_adhA(digested with XbaI, PstI)

adhA

Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)

Annealing temperature: 65 °C
Bands as expected (~ 1200 bp)

Liquid culture for a restriction digest was prepared.
Plasmid isolation of pSB1A2-T7_adhA
Restriction digestion with EcoRI and PstI

Bands as expected (backbone: ~ 2200 bp and insert: ~ 1300 bp)

Successful sequencing
For the protein expression analysis of AdhA we made a cultivation. Samples were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced when the culture reached a OD₆₀₀ 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples one, two and three and 20 hours later. Of these samples, we made a SDS Page

SDS page from pSB1A2_T7_adhA
The sizes of the AdhA is ~ 35 Da

GC-MS analysis of the isobutanol production

For the analysis of the isobutanol production we decided to use a GC-MS analysis. Because we will cultivate in LB medium, we made a calibration line with LB and the following concentrations of isobutanol for further analysis and quantification of our samples.

0,5 % isobutanol
0,1 % isobutanol
0,05 % isobutanol
0,01 % isobutanol
0,001 % isobutanol

Solvent extraction with 100µl LB-sample and 900µl aceton
5 min centrifugation
150 µl supernatant in the GC-MS vials for the analysis

The detection of isobutanol works for all tested concentrations but the 2-butanol peak was to high, so we need to do this again with 0.1% 2-Butanol.

@@ Line 40: / Line 40: @@
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@@ Line 49: / Line 49: @@
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+<ul>
+	<li><b><i>adhA</i></b></li>
+	<ul><li>This week we tried to verify the positive cutures that were identified last week by a restriction digest</li>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1K3_<i>adhA</i></li>
+</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> of pSB1K3_<i>adhA</i> </li>
+	<ul>
+		<li>Bands as expected (~1200 bp)</li>
+                <div class="element" style="height:350px; width:200px; text-align:center">
+                              <a href="https://static.igem.org/mediawiki/2014/9/9a/Bielefeld_CeBiTec_2014-10-14_adhA_Kontrollverdau_09_01.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/9a/Bielefeld_CeBiTec_2014-10-14_adhA_Kontrollverdau_09_01.png" height="230px"></a><br><font size="2">Agarose gel from the restriction digestion of pSB1K3_<i>adhA</i> with <i>EcoR</i>I and <i>Pst</i>I. As a Ladder we used the <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder. The fragments in both lines have the right sizes.</a>. </font>
+                        </div>
+ </ul>	</ul><li>Successful sequencing</li></ul></ul><ul>
+<li><b><i>T7_alsS_ilvC_ilvD_kivD</i></b></li>
+	<ul><li>This week we tried to verify the positive cutures that were identified last week by a restriction digest</li>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_T7_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> of pSB1A2_T7_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> </li>
+	<ul>
+		<li>Bands not as expected (Backbone: ~ 2200 and insert: ~ 7500bp)</li>
+ </ul>	</ul></ul></ul></ul>
+<ul>
+	<li><b><i>pSB1K3_alsS_ilvC_ilcD_kivD_adhA</i></b></li>
+	<ul>
+		<li>This week we tried to combine the pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> with <i>adhA</i></li>
+                       <ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
+	<ul>
+		<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li>pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+		</ul>
+		<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i></i></li>
+		</ul>
+	</ul>
+</ul><ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-ilvD-kivD" target="_blank">fw-ilvD-kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-pSB1C3-adhA" target="_blank">rev-pSB1C3-adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands not as expected (~ 3800 bp)</li>
+	</ul>
+</ul>
+	</ul>
+</ul>
+<br>
           </div>
        </div>
@@ Line 56: / Line 115: @@
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@@ Line 65: / Line 124: @@
              </div>
              <div class="content" style="margin-right:10%; margin-left:10%">
+<ul>
+	<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
+	<ul>
+		<li>This week we tried to reclone <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> into pSB1C3 </li>
+                 <ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> and pSB1C3_<i>RFP</i> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
+	<ul>
+		<li>Bands as expected <br>(~ 7500 bp for <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> and ~ 2200 bp for pSB1C3)</li>
+	</ul><li>Clean up and Ligation as explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a></li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+	</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands not as expected (~ 1800 bp)</li>
+	</ul>
+</ul>
+</ul>
+<ul>
+	<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i></i></b></li>
+	<ul>
+		<li>This week we tried to contruct the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i>-Plasmid by <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li>
+                       <ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li>
+	<ul>
+		<li>Upstream part pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
+		<ul>
+			<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+		</ul>
+		<li>Downstream part pSB1K3_<i>adhA</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>adhA</i></li>
+		</ul>
+                                   <li>Destination part pSB1C3_RFP (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+		</ul>
+	</ul>
+</ul><ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD </a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65°C</li>
+		<li>Bands not as expected (~ 3000 bp)</li>
+	</ul>
+</ul>
+	</ul>
+</ul></ul>
+<br>
           </div>
        </div>
@@ Line 72: / Line 185: @@
 <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
-   <div id="text">
+   <div id="text" style="text-align:left">
 <div class="tab" id="Week3">
              <div class="show">
@@ Line 81: / Line 194: @@
              </div>
              <div class="content" style="margin-right:10%; margin-left:10%">
+<ul>
+	<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
+	<ul>
+		<li>This week we tried to reclone <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> into pSB1C3 </li>
+                 <ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> and pSB1C3_<i>RFP</i> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
+	<ul>
+		<li>Bands as expected <br>(~ 7500 bp for <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> and ~ 2200 bp for pSB1C3)</li>
+	</ul><li>Clean up and Ligation as explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a></li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+	</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands not as expected (~ 1800 bp)</li>
+	</ul>
+</ul></u></ul></ul>
+<ul>
+	<li><b><i>pSB1C3_<i>adhA</i></b></li>
+	<ul>
+		<li>This week we tried to reclone <i>adhA</i> into pSB1C3 </li>
+                 <ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of pSB1K3_<i>adhA</i></i> and pSB1C3_<i>RFP</i> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
+	<ul>
+		<li>Bands as expected <br>(~ 1200 bp for <i>adhA</i> and ~ 2200 bp for pSB1C3)</li>
+	</ul><li>Clean up and Ligation as explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a></li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+	</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands not as expected (~ 1200 bp)</li>
+	</ul></ul>
+</ul>
+<ul>
+	<li><b><i>pSB1A2_T7_<i>adhA</i></i></b></li>
+	<ul>
+		<li>This week we tried to contruct the pSB1A2_T7_<i>adhA</i>-Plasmid </li>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
+	<ul>
+		<li>Backbone pSB1A2_T7(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li>pSB1A2_T7</li>
+		</ul>
+		<li>Insert pSB1K3_<i>adhA</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>adhA</i></li>
+		</ul>
+	</ul>
+</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands not as expected (~ 1200 bp)</li>
+	</ul></ul>
+</ul>
+	</ul>
+</ul>
+<ul>
+	<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i></i></b></li>
+	<ul>
+		<li>This week we tried to contruct the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i>-Plasmid by <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li>
+                       <ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li>
+	<ul>
+		<li>Upstream part pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
+		<ul>
+			<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+		</ul>
+		<li>Downstream part pSB1K3_<i>adhA</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>adhA</i></li>
+		</ul>
+                                   <li>Destination part pSB1C3_RFP (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+		</ul>
+	</ul>
+</ul><ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD </a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65°C</li>
+		<li>Bands not as expected (~ 3000 bp)</li>
+	</ul>
+</ul>
+<ul><li>Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a>. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. Thereby we discovered that the pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> has an illigal restriction side. <br> Because of the integration of RBS's between all genes in the <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>-Plasmid, a restriction side between <i>alsS</i> and a RBS occurs.<br>New primers were ordered: <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a></li></ul>
+	</ul>
+</ul>
           </div>
        </div>
@@ Line 88: / Line 301: @@
 <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
-   <div id="text">
+   <div id="text" style="text-align:left">
 <div class="tab" id="Week4">
              <div class="show">
@@ Line 97: / Line 310: @@
              </div>
              <div class="content" style="margin-right:10%; margin-left:10%">
+<ul>
+	<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
+	<ul>
+		<li>This week the new primers <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a> arrived. We tried to remove the illegale restriction side.</li>
+                     <ul>
+	<li>We tried different <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a>s to increase the chance to get a correct BioBrick. Therefore we amplified five parts for two combination possibilities in the Gibson Assembly:
+<ol><li>  <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a> on pSB1C3_RFP <br>&#8594; pSB1C3 with ~ 2200 bp</li>
+<li>  <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primerfw_pSB1C3_alsS" target="_blank">fw_pSB1C3_alsS</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a> on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>alsS</i> with ~ 1800 bp</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a> on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> with ~ 5200 bp </li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a> on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>ilvC</i>_<i>ilvD</i> with ~ 3600 bp</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>  on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>kivD</i> with ~ 1800 bp</li></ol>
+	<ul>
+		<li>Annealing temperature: 65 &deg;C</li>
+		<li>Bands as expected (see above)</li>
+	</ul>
+</ul>
+<ul>
+	<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
+</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with the previous PCR products:  <ul>
+	<li>1., 2. and 3.</li><li>1., 2., 4. and 5. </li>
+</ul></li>
+</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primerfw_pSB1C3_alsS" target="_blank">fw_pSB1C3_alsS</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands as expected (~ 1800 bp)</li>
+	</ul>
+</ul>
+<li>Liquid cultures for a restriction digest were prepared.</li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>alsS</i>-<i>ilvC</i>-<i>ilvD</i>-<i>kivD</i> without illigale restriction side</li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
+	               <ul>
+	                  <li>Bands as expected (backbone: 2,2 kb and insert: 7 kb)</li>
+	               </ul>
+<li>Successful sequencing</li>
+	</ul>
+</ul>
+<ul>
+	<li><b><i>pSB1C3_adhA</i></b></li>
+	<ul>
+		<li>This week we tried to construct the pSB1C3_<i>adhA</i> using Gibson Assembly.</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the pSB1C3_RFP for pSB1C3 as backbone (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_adhA_pSB1C3" target="_blank">fw_adhA_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_adhA_pSB1C3" target="_blank">rev_adhA_pSB1C3</a>) and pSB1K3_<i>adhA</i> for <i>adhA</i> as insert (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>)  </li>
+	<ul>
+		<li>Annealing temperature: 65 &deg;C</li>
+		<li>Bands as expected (~ 1200 bp for the <i>adhA</i> and ~ 2200 bp for the backbone)</li>
+	</ul>
+<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>adhA</i> and pSB1C3</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands as expected (~ 1200 bp)</li>
+	</ul>
+<li>Liquid cultures for a restriction digest were prepared.</li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>adhA</i></li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
+	               <ul>
+	                  <li>Bands as expected (backbone: 2,2 kb and insert: 1,2 kb)</li>
+	               </ul>
+<li>Successful sequencing</li>
+	</ul>
+</ul>
           </div>
        </div>
@@ Line 104: / Line 399: @@
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-   <div id="text">
+   <div id="text" style="text-align:left">
 <div class="tab" id="Week5">
              <div class="show">
@@ Line 113: / Line 408: @@
              </div>
              <div class="content" style="margin-right:10%; margin-left:10%">
+<ul>
+	<li><b><i>pSB1C3_ptac_alsS_ilvC_ilcD_kivD</i></b></li>
+	<ul>
+		<li>This week we tried to combine the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> contruct (without illegale restriction side) with the <i>ptac</i> promotor.</li>
+             	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix and Prefix)</li>
+	<ul>
+		<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li>pSB1C3_<i>ptac</i></li>
+		</ul>
+		<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></li>
+		</ul>
+               AND:
+               <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>)</li>
+		<ul>
+			<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_pSB1C3</li>
+		</ul>
+		<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
+		<ul>
+			<li><i><i>ptac</i></i></li>
+		</ul>
+	</ul>
+          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 55°C</li>
+		<li>Bands as expected (~ 3000 bp)</li>
+	</ul>
+<li>Liquid culture for a restriction digest was prepared.</li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>ptac</i>_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
+	               <ul>
+	                  <li>Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)</li>
+	               </ul>
+<li>Successful sequencing</li>
+	</ul>
+</ul>
+<ul>
+	<li><b><i>pSB1C3_ptac_adhA</i></b></li>
+	<ul>
+		<li>This week we tried to combine the pSB1C3_<i>adhA</i> contruct with the <i>ptac</i> promotor.</li>
+             	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix and Prefix)</li>
+	<ul>
+		<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li>pSB1C3_<i>ptac</i></li>
+		</ul>
+		<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>adhA</i></li>
+		</ul>
+               AND:
+               <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>)</li>
+		<ul>
+			<li><i>adhA</i>_pSB1C3</li>
+		</ul>
+		<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
+		<ul>
+			<li><i>ptac</i></li>
+		</ul>
+	</ul>
+          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 55°C</li>
+		<li>Bands as expected (~ 2800 bp)</li>
+	</ul>
+<li>Liquid cultures for a restriction digest were prepared.</li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>adhA</i></li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
+	               <ul>
+	                  <li>Bands as expected (backbone: ~ 2200 kb and insert: ~ 2400 bp)</li>
+	               </ul>
+<li>Successful sequencing</li>
+	</ul>
+</ul>
+<ul>
+	<li><b><i>pSB1C3_alsS_ilvC_ilcD_kivD_adhA</i></b></li>
+	<ul>
+		<li>This week we tried to combine the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> contruct (without illegale restriction side) with the <i>adhA</i> from <i>L. lactis</i>.</li>
+             	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix and Prefix)</li>
+	<ul>
+		<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
+		<ul>
+			<li>pSB1C3_<i>adhA</i></li>
+		</ul>
+		<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></li>
+		</ul>
+               AND:
+               <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
+		</ul>
+		<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i><i>ptac</i></i></li>
+		</ul>
+	</ul>
+          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
+<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 55°C</li>
+		<li>Bands as expected (~ 3000 bp)</li>
+	</ul>
+<li>Liquid culture for a restriction digest was prepared.</li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i></li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
+	               <ul>
+	                  <li>Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)</li>
+	               </ul>
+<li>Successful sequencing</li>
+	</ul>
+</ul>
+<ul>
+	<li><b><i>pSB1A2_T7_<i>adhA</i></i></b></li>
+	<ul>
+		<li>This week we tried to contruct the pSB1A2_T7_<i>adhA</i>-Plasmid </li>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
+	<ul>
+		<li>Backbone pSB1A2_T7(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li>pSB1A2_T7</li>
+		</ul>
+		<li>Insert pSB1C3_<i>adhA</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
+		<ul>
+			<li><i>adhA</i></li>
+		</ul>
+	</ul>
+</ul>
+<ul>
+	<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 65 °C</li>
+		<li>Bands as expected (~ 1200 bp)</li>
+	</ul>
+<li>Liquid culture for a restriction digest was prepared.</li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2-<i>T7</i>_<i>adhA</i></li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
+	               <ul>
+	                  <li>Bands as expected (backbone: ~ 2200 bp and insert: ~ 1300 bp)</li>
+	               </ul>
+<li>Successful sequencing</li>
+<li>For the protein expression analysis of AdhA we made a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation for Expression of recombinant proteins" target="_blank">cultivation</a>. Samples were taken like explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">cell lysis for a SDS-PAGE Protocol</a>. Protein expression was induced when the culture reached a OD<sub>600</sub> 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples one, two and three and 20 hours later. Of these samples, we made a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS Page</a>
+<div class="element" style="height:300px; width:450px; text-align:center">
+                      <a href="https://static.igem.org/mediawiki/2014/5/53/Bielefeld-CeBiTec_14-10-16_SDS_T7_adhA.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/5/53/Bielefeld-CeBiTec_14-10-16_SDS_T7_adhA.jpg" height="230px"></a><br><font size="1">SDS page from pSB1A2_<i>T7</i>_<i>adhA</i>
+<br>The sizes of the AdhA is ~ 35 Da</font>
+                    </div>
+</li>
+</ul>
+</ul>
+<ul>
+	<li><b><i>GC-MS analysis of the isobutanol production</i></b></li>
+	<ul>
+		<li>For the analysis of the isobutanol production we decided to use a GC-MS analysis. Because we will cultivate in LB medium, we made a calibration line with LB and the following concentrations of isobutanol for further analysis and quantification of our samples.</li>
+              <ul>
+                  <li>0,5 % isobutanol</li>
+                  <li>0,1 % isobutanol</li>
+                  <li>0,05 % isobutanol</li>
+                  <li>0,01 % isobutanol</li>
+                  <li>0,001 % isobutanol</li>
+              </ul>
+Additionally we prepared GC-grade aceton with 1% 2-Butanol as an internal standard. >br>The samples were treated as described below:
+<ul><li>Solvent extraction with 100&#181;l LB-sample and 900&#181;l aceton</li>
+<li>5 min centrifugation</li>
+<li>150 &#181;l supernatant in the GC-MS vials for the analysis </li></ul> </li>
+        <li>The detection of isobutanol works for all tested concentrations but the 2-butanol peak was to high, so we need to do this again with 0.1% 2-Butanol.</li>
+	</ul>
+</ul>
           </div>
        </div>

Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Sep