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August

rMFC

CO₂ fixation

Isobutanol

Biosafety

General

Week 1 08/04 - 08/10

Promoters T7, p_tac and p_Tet (BBa_K1465213 (pSB1C3_p_tac_Hneap), BBa_K1465212 (pSB1C3_p_tac))

We tried to assemble some inserts with three different promoters to test which one is the best choice.

Plasmid isolation of p_tac, p_tac, T7, prkA, Hneap, sRNA:pfkA and can
BioBrick Assembly (Suffix)

Backbones (digested with SpeI, PstI)

pSB1A2_T7
pSB1C3_p_tac
pSB1K3_p_Tet

Inserts (digested with XbaI, PstI)
- prkA
- Hneap
- sRNA:pfkA

Transformation of all constructs with electrocompotetent cells
Colony PCR of pSB1C3_p_tac_prkA, pSB1C3_p_tac_Hneap and pSB1C3_p_tac_sRNA:pfkA (VF-Primer, VR-Primer)

Annealing temperature: 55 °C
Bands as expected (~2500 bp (p_tac_prkA), ~3200 bp (p_tac_Hneap), ~1700 bp (p_tac_sRNA:pfkA))

Colony PCR of pSB1A2_T7_prkA, pSB1A2_T7_Hneap and pSB1A2_T7_sRNA:pfkA (VF-Primer, VR-Primer)

Annealing temperature: 55 °C
Bands not as expected

transform

Colony PCR of pSB1K3_p_Tet_prkA, pSB1K3__Tet_Hneap and pSB1K3__Tet_sRNA:pfkA (VF-Primer, VR-Primer)

Annealing temperature: 55 °C
Bands not as expected

Colony PCR of pSB1A2_T7 from the 2013 distribution plate (VF-Primer, VR-Primer)

Annealing temperature: 55 °C
Bands as expected (~300 bp)

Plasmid isolation of pSB1C3_p_tac_prkA, pSB1C3_p_tac_Hneap, pSB1C3_p_tac_sRNA_pfkA and pSB1A2_T7

csoS1-4 (shell proteins csoS4AB and csoS1CAB)

We used the amplified products to assemble and transform them to get the shell protein construct.

Restriction digestion with DpnI
Gibson Assembly with csoS1-4 and pSB1C3
Transformation with electrocompotetent cells
Colony PCR (VF-Primer, VR-Primer)

Annealing temperature: 55 °C
Bands as expected (~2100 bp)

Plasmid isolation
Restriction digestion with SpeI and XbaI

Bands as expected (~1800 bp and ~2200 bp)

Agarose gel from the restriction digestion with SpeI and XbaI. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

can and csoS1-4

We tried to assemble the shell proteins and the carbonic anhydrase for the carboxysome.

BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

pSB1C3_can

Insert (digested with XbaI, PstI)
- csoS1-4

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

Annealing temperature: 55 °C
Bands as expected (~3600 bp)

Plasmid isolation of pSB1C3_can_csoS1-4

can

glpX

We tried to assemble and transform the glpX parts again.

Restriction digestion with DpnI
Gibson Assembly with glpX_1, glpX_2 and pSB1K3
Transformation with electrocompotetent cells
Colony PCR on one part of the fragment (fw-pSB1C3-BBa_B0034-SBPase, rv-SBPase-Schnittstelle)

Annealing temperature: 55 °C
Bands as expected (~500 bp)

Plasmid isolation of glpX

prkA and pHnCBscoS1D

We tried to amplify prkA again and to assemble it with the plasmid pHnCBcsoS1D.

Plasmid isolation of DH5α prkA
PCR amplification on the isolated prkA (prkA_pHn_fwd, prkA_pHn_rev)

Annealing temperature: 55 °C
Bands as expected (~1000 bp)

PCR products were purified out of the gel
Gibson Assembly with prkA and pHnCBcsoS1D

RuBisCO

We tried to assemble both RuBisCO with pSB1C3_p_tac_prkA

BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

p_tac_prkA

Inserts (digested with XbaI, PstI)
- Hneap
- SELAN
We assembled pSB1C3_p_tac_prkA with Hneap respectively SELAN

Colony PCR (VF-Primer, VR-Primer)

Annealing temperature: 55 °C

pSB1C3_p_tac_prkA_Hneap

Bands not as expected (too short).

_tac

Hneap

pSB1C3_p_tac_prkA_SELAN

Bands as expected (~4200 bp)

Plasmid isolation of p_tac_prkA_Hneap and p_tac_prkA_SELAN

Selan

_tac

can_csoS1-4 and csoS1D

We tried to assemble pSB1C3_can_csoS1-4 and csoS1D and transform the construct.

BioBrick Assembly Suffix:

Backbone (digested with SpeI,PstI)

pSB1C3_can_csoS1-4

Insert (digested with XbaI, PstI)

csoS1D

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

Annealing temperature: 55 °C
Bands as expected (~4300 bp)

Week 2 08/11 - 08/17

glpX

This week we tried to amplify the backbone pSB1C3 for glpX again. We took the pSB1C3_RFP as a template.

PCR amplification of the pSB1C3_RFP backbone (fw_SBPase_pSB1C3, rv_SBPase_pSB1)

Annealing temperature: 54 °C
Bands as expected (~2100 bp)

PCR products were purified
Restriction digestion with DpnI
Gibson Assembly with glpX_1, glpX_2 and pSB1C3
Transformation with electrocompotetent cells

Colony PCR (VF-Primer, VR-Primer)

Annealing temperature: 55 °C
Bands as expected (~1300 bp)

Plasmid isolation of pSB1C3_glpX

csoS1-4

We tried to isolate the right plasmid again.

Plasmid isolation of pSB1C3_csoS1-4
Restriction digestion with PstI and EcoRI as a control

Bands as expected (~2100 bp (backbone) and ~1700 bp (insert))

can (BBa_K1465205)

can

Purification of the carboxysome

We want to express and purify the carboxysome based on the plasmid pHnCBcsoS1D, which we received from addgene. The procedure is based on methods described by Bonacci et al., 2011 and So et al., 2004.

Grow cells containing the pHnCBcsoS1D in TB medium containing antibiotic at 20 °C overnight
Dilute the overnight culture 1:50 in TB medium containing antibiotic. A culture volume of 500 ml was used
Grow cells until they reach an OD₆₀₀ of 0.9-1.2
Induce protein expression by adding IPTG to a final concentration of 50 µM. Grow cells overnight
Harvest cells by centrifugation with 4500 x g for 20 min at 4 °C
Resuspend cells in 50 mL TEMB buffer. Use protease inhibitors, for example PMSF
- 5 mM Tris-HCl (pH 8.0)
- 1 mM EDTA
- 10 mM MgCl₂
- 20 mM NaHCO₃
Cell lysis via sonification (6 x 1 min with 1 min cooling interval between every cycle). Therefore a Bandelin Sonopuls HD 2070 with a SH70G with a power of 70 W was used. The power was adjust to 70 %
Centrifuge the disruptet cell material with 12,000 x g for 30 min at 4 °C to spin down cell debris. The cellular proteins should be in the supernatant.
Collect the supernatant and centrifuge with 40,000 x g for 30 min at 4 °C
Discard supernatant. Resuspend the resulting pellet in 20 mL of a 33 % (vol/vol) solution of CellLytic B (Sigma) in TEMB
Centrifuge again with 40,000 x g for 30 min at 4 °C
Discard supernatant. Resuspend pellet in 3 mL TEMB buffer. Clarify by centrifugation with 3,000 x g for 1 min
Load the supernatant onto 10 to 50 % (wt/vol) linear sucrose gradient. Sucrose gradients are made by slowly dribbling high % to low % sucrose down the side of the tube. For ultracentrifugation Polyallomer tubes with a volume of 13,2 mL were used. Ultraspin the sample in a Beckman Coulter Optima^TM LE 80 K Ultracentrifuge with a SW41 Ti Swinging Rotor Bucket with 105,000 x g for 30 min
After centrifugation, the carboxysome should be seen as a band near the middle of the gradient

Week 3 08/18 - 08/24

pHnCBcsoS1D_prkA

This week we tried to transform the pHnCBcsoS1D_prkA construct.

Transformation with electrocompotetent cells
Colony PCR (prkA_pHn_rev, prkA_pHn_fwd)

Annealing temperature: 55°C
Bands not as expected

csoS1-4

This week we tried to amplify and transform the shell proteins again.

PCR amplification (fw_pSB1C3_csoS4A, rv_csoS4A_PstI)

Annealing temperature: 55 °C
Bands as expected (~180 bp)

PCR amplification (fw_PstI_csoS4A, rv_SpeI_Intergen)

Annealing temperature: 55 °C
Bands as expected (~1200 bp)

PCR amplification (fw_SpeI_Intergen, rv_csoS1_pSB1C3)

Annealing temperature: 55 °C
Bands as expected (~350 bp)

PCR amplification of the backbone (fw_csoS1_pSB1C3, rv_pSB1C3_csoS4A)

Annealing temperature: 55 °C
Bands as expected (~2070 bp)

Agarose gel from PCR. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

All PCR products were purified
Restriction digestion with DpnI
Gibson Assembly with csoS1-4 and pSB1C3
Transformation with electrocompotetent cells

prkA and p_Tet

This week we tried to assemble the p_Tet promoter with the prkA.

BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

pSB1K3_p_Tet

Insert (digested with XbaI, PstI)

prkA

Transformation with electrocompotetent cells

prkA

Colony PCR (VF-Primer, VR-Primer)

Annealing temperature: 55 °C
Bands as expected (~2200 bp)

Agarose gel from colony PCR. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

Hneap and p_Tet

This week we tried to assemble the p_Tet promoter with the Hneap.

BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

pSB1K3_p_Tet

Insert (digested with XbaI, PstI)

Hneap

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

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

Agarose gel from colony PCR. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

sap_2

This week we tried to bring the synthesized sap_2 construct in the pJet vector with blunt end cloning. We also amplified the backbonefor an assembly.

Blunt-End cloning of sap_2 in the pJet vector
Colony PCR (Primer of pJet set)

Annealing temperature: 55 °C
Bands as expected (~1500 bp)

Agarose gel from colony PCR. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

PCR amplification of the backbone (pSB1C3) (pSB1C3_pre_sap_1, pSB1C3_suf_sap2)

Annealing temperature: 55 °C
Bands as expected (~2100 bp)

Agarose gel from colony PCR. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

GFP BioBrick (BBa_E0040)

This week we tried isolate the green fluorescent protein GFP (pSB1A2_GFP) of the parts distribution 2014.

Plasmid isolation of pSB1A2_GFP

Purification vector

This week we tried to amplify the T7_RBS promoter for the purification vector. Additionally we amplified the intein tag containing a chitin binding domain.

PCR amplification (RBS_int_rev, pSB1C3_int_fw) of the promoter (T7_RBS)

Annealing temperature: 55°C
Bands as expected (~2000 bp)

PCR amplification (int_RBS_fw, int_pSB1C3_rev) the intein tag with chitin binding domain (intCBD)

Annealing temperature: 55°C
Bands as expected (~1000 bp)

PCR products were purified
Gibson Assembly with T7_RBS and intCBD
Transformation with electrocompotetent cells
Colony PCR (VF-Primer, VR-Primer)

Annealing temperature: 55°C
Bands as expected (~3300 bp)

Plasmid isolation of pSB1C3_T7_RBS_intCBD

Purification of the carboxysome

As the purification of the carboxysome showed no results, we decided to induce protein expression with higher concentrations of IPTG.

Cultivation was carried out using a modified method of Cultivation for Expression of recombinant proteins . Cells were continous cultivated by a temperature of 20 °C, and protein expression was induced when the culture reaches an OD₆₀₀ of 0.9 - 1.2. IPTG in different final concentrations of 0.5 mM, 2 mM and 5 mM was used to verify the expression of the carboxysomal proteins through SDS-PAGE and MALDI-TOF . Samples were generated using the protocol for Fast Cell Lysis for SDS-PAGE.

Proteinexpression of pHnCBcsoS1D
induced with 0.5 mM IPTG

Proteinexpression of pHnCBcsoS1D
induced with 2 mM IPTG

Proteinexpression of pHnCBcsoS1D
induced with 5 mM IPTG

Week 4 08/25 - 08/31

sap_2

We tried to isolate pJet_sap_2 for further experiments.

Plasmid isolation of pJet_sap_2

glpX

We tried to amplify the pSB1K3 backbone for an assembly with glpX.

PCR amplification of the pSB1C3_RFP backbone (fw_SBPase_pSB1C3, rv_SBPase_pSB1)

Annealing temperature: 54 °C
Bands as expected (~2100 bp)

PCR products were purified
Restriction digestion with DpnI
Gibson Assembly with glpX_1, glpX_2 and pSB1K3
Transformation with electrocompotetent cells

csoS1-4 (BBa_K1465206)

We tried to find correct colonies of the psB1C3_csoS1-4 construct so another part of the carboxysome is complete.

Colony PCR (fw_pSB1C3_csoS4A, rv_csoS1_pSB1C3)

Annealing temperature: 65 °C
Bands (not) as expected (~1600 bp)

Agarose gel from colony PCR. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

Plasmid isolation of pSB1C3_csoS1-4
Restriction digestion with EcoRI and PstI

Bands as expected (~2000 bp backbone, ~1800 bp insert)

sRNA:pfkA and p_tac

We tried to assemble the sRNA:pfkA construct and pSB1C3_p_tac

BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

pSB1C3_ptac

Insert (digested with XbaI, PstI)

sRNA:pfkA

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

Annealing temperature: 55 °C
Bands as expected (~1700 bp)

Plasmid isolation of pSB1C3_p_tac_sRNA:pfkA

sRNA:pfkA and T7 (BBa_K1465227)

We tried to assemble the sRNA:pfkA construct and pSB1A2_T7

BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

pSB1A2_T7

Insert (digested with XbaI, PstI)

sRNA:pfkA

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

Annealing temperature: 55 °C
Bands as expected (~900 bp)

Plasmid isolation of pSB1A2_T7_sRNA:pfkA
BioBrick Assembly (Suffix)

Backbone (digested with SpeI, PstI)

pSB1C3

Insert (digested with XbaI, PstI)

T7_sRNA:pfkA

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

Annealing temperature: 55 °C
Bands as expected (~600 bp)

Agarose gel from colony PCR. As a Ladder we used GeneRuler™ 1 kb DNA Ladder from Thermo Scientific.

Plasmid isolation of pSB1C3_T7_sRNA:pfkA

GFP

We tried to amplify GFP for a fusion with the shell proteins.

PCR amplification (fw-csoS1A-GFP, rv-csoS1B-GFP)

Annealing temperature: 54 °C
Bands as expected (~750 bp)

PCR products were purified out of the gel

can and csoS1-4

We tried to assemble can and csoS1-4 for the carboxysome.
- BioBrick Assembly (Prefix)
- Transformation with electrocompotetent cells

Purification of the carboxysome

The results of our experiments suggest that higher IPTG concentrations are needed for a efffective protein expression of the carboxysome. For this reason, we induced the protein expression with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal , but slightly modificated. The culture volumen was upscaled to 1 litre. For a more effective cell lysis, the sonification protocol was modified using eight cylces a one min with one min cooling intervals for sonification.

@@ Line 50: / Line 50: @@
               <div class="content"  style="margin-right:10%; margin-left:10%">
                  <ul> <!-- gesamte Liste -->
-<li><b>Promotors T7, p<sub>tac</sub> and p<sub>Tet</sub> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465213" target="_blank">(BBa_K1465213 (pSB1C3_p<sub>tac</sub>_Hneap)</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465212" target="_blank">BBa_K1465212 (pSB1C3_p<sub>tac</sub>))</a></b></li>
+<li><b>Promoters T7, p<sub>tac</sub> and p<sub>Tet</sub> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465213" target="_blank">(BBa_K1465213 (pSB1C3_p<sub>tac</sub>_Hneap)</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465212" target="_blank">BBa_K1465212 (pSB1C3_p<sub>tac</sub>))</a></b></li>
                    <ul> <!-- Promotors -->
-                     <li>We tried to assemble some inserts with three different promotors to test which one is the best  choice.</li>
+                     <li>We tried to assemble some inserts with three different promoters to test which one is the best  choice.</li>
                      <ul>
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of p<sub>tac</sub>, p<sub>tac</sub>, T7, <i>prkA, Hneap, sRNA:pfkA</i> and <i> can </i></li>
@@ Line 86: / Line 86: @@
 		        <li>Bands not as expected</li>
                          &rarr; Showed in all cases a band 400 bp over the expected value. We tried to extract and
-                         <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">transform</a> the promotor from another distribution plate (2013)
+                         <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">transform</a> the promoter from another distribution plate (2013)
 	              </ul>
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of pSB1K3_p<sub>Tet</sub>_prkA, pSB1K3_<sub>Tet</sub>_Hneap
@@ Line 345: / Line 345: @@
 <br>
-<li><b><i>Purification of the carboxysome</b></i></li>
+<li><b>Purification of the carboxysome</b></li>
                   <ul>
-                     <li>We want to express and purify the carboxysome based on the plasmid pHnCBcsoS1D, which we received from addgene. The procedure is based on methods described by <a href="#bonacci2011">Bonacci et al., 2011</a> and <a href="#so2004">So et al., 2004</a>; </li>
+                     <li>We want to express and purify the carboxysome based on the plasmid pHnCBcsoS1D, which we received from addgene. The procedure is based on methods described by <a href="http://www.pnas.org/content/109/2/478" target="_blank"">Bonacci et al., 2011</a> and <a href="#http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321498/pdf/1109.pdf">So et al., 2004</a>. </li>
                      <ul>
                         <li>Grow cells containing the pHnCBcsoS1D in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#TBmedium" target="_blank">TB medium </a> containing antibiotic at 20 °C overnight</li>
@@ Line 354: / Line 354: @@
 <li> Induce protein expression by adding IPTG to a final concentration of 50 µM. Grow cells overnight </li>
 <li> Harvest cells by centrifugation with 4500 x g for 20 min at 4 °C </li>
-<li> Resuspend cells in 50 mL TEMB buffer. Use protease inhibitors, for example PMSF.
+<li> Resuspend cells in 50 mL TEMB buffer. Use protease inhibitors, for example PMSF
 	               <ul>
 	           	   TEMB buffer:
@@ Line 362: / Line 362: @@
 <li> 20 mM NaHCO<sub>3</sub> </li>
 	               </ul>
-<li> Cell lysis via sonification (6 x 1 min with 1 min cooling interval between every cycle). Therefore a (<a href="http://www.sigmaaldrich.com/germany.html" target="_blank">Bandelin</a> Sonopuls HD 2070 with a SH70G with a power of 79 W was used. The power was adjust to 70 % </li>
+<li> Cell lysis via sonification (6 x 1 min with 1 min cooling interval between every cycle). Therefore a <a href="http://www.sigmaaldrich.com/germany.html" target="_blank">Bandelin</a> Sonopuls HD 2070 with a SH70G with a power of 70 W was used. The power was adjust to 70&nbsp;% </li>
 <li> Centrifuge the disruptet cell material with 12,000 x g for 30 min at 4 °C to spin down cell debris. The cellular proteins should be in the supernatant. </li>
 <li>Collect the supernatant and centrifuge with 40,000 x g for 30 min at 4 °C </li>
@@ Line 368: / Line 368: @@
 <li> Centrifuge again with 40,000 x g for 30 min at 4 °C </li>
 <li> Discard supernatant. Resuspend pellet in 3 mL TEMB buffer. Clarify by centrifugation with 3,000 x g for 1 min </li>
-<Load the supernatant onto 10 to 50 % (wt/vol) linear sucrose gradient. Sucrose gradients are made by slowly dribbling high % to low % sucrose down the side of the tube. Ultraspin the sample in with 105,000 x g for 30 min </li>
+<li> Load the supernatant onto 10 to 50 % (wt/vol) linear sucrose gradient. Sucrose gradients are made by slowly dribbling high % to low % sucrose down the side of the tube. For ultracentrifugation Polyallomer tubes with a volume of 13,2 mL were used. Ultraspin the sample in a Beckman Coulter Optima<sup>TM</sup> LE 80 K Ultracentrifuge with a SW41 Ti Swinging Rotor Bucket with 105,000 x g for 30 min </li>
-<li> After centrifugation, the carboxysome should be seen as a band near the middle of the gradient. </li>
+<li> After centrifugation, the carboxysome should be seen as a band near the middle of the gradient </li>
-&rarr; The purification was not succesful, as you could not recognize a visible band in the gradient.
+&rarr; The purification was not succesful, as you could not recognize a visible band in the gradient
                      </ul>
                   </ul>
@@ Line 401: / Line 398: @@
                      <ul>
                         <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
-                        <font color="red"><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#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)
+                        <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#prkA_pHn_rev" target="_blank">prkA_pHn_rev</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#prkA_pHn_fwd" target="_blank">prkA_pHn_fwd</a>)
                         </li>
 	               <ul>
-		         <li>Annealing temperature: ...</li>
+		         <li>Annealing temperature: 55°C </li>
-		         <li>Bands (not) as expected (~... bp)</li>
+		         <li>Bands not as expected</li>
-	               </ul></font>
+                         &rarr; We got a lot of bands and could not interpret the result.
+	               </ul>
                      </ul>
@@ Line 469: / Line 467: @@
                   <li><b><i>prkA</i> and <i>p<sub>Tet</i></b></li>
 	         <ul>
-	 	   <li>This week we tried to assemble the <i>p<sub>Tet</sub></i> promotor with the <i>prkA</i>.</li>
+	 	   <li>This week we tried to assemble the <i>p<sub>Tet</sub></i> promoter with the <i>prkA</i>.</li>
                     <ul>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
@@ Line 501: / Line 499: @@
 <li><b><i>Hneap</i> and <i>p<sub>Tet</i></b></li>
 	         <ul>
-	 	   <li>This week we tried to assemble the <i>p<sub>Tet</sub></i> promotor with the <i>Hneap</i>.</li>
+	 	   <li>This week we tried to assemble the <i>p<sub>Tet</sub></i> promoter with the <i>Hneap</i>.</li>
                     <ul>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
@@ Line 534: / Line 532: @@
                        <ul>
                          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BluntEnd" target="_blank">Blunt-End</a> cloning of <i>sap_2</i> in the pJet vector</li>
-                         <li><font color="red"> <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#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)
+                         <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/Protocols#BluntEnd" target="_blank">Primer of pJet set</a>)
                          </li>
 	                <ul>
-		          <li>Annealing temperature: ... °C</li>
+		          <li>Annealing temperature: 55 °C</li>
-		          <li>Bands as expected (~1500 bp)</li></font>
+		          <li>Bands as expected (~1500 bp)</li>
                            <div class="element" style="height:350px; width:120px; text-align:center">
                              <a href="https://static.igem.org/mediawiki/2014/0/06/Bielefeld_CeBiTec_2014-09-25_sap_2_cPCR%28pJet%29_08_21.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/0/06/Bielefeld_CeBiTec_2014-09-25_sap_2_cPCR%28pJet%29_08_21.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. As a Ladder we used <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 from Thermo Scientific</a>. </font>
@@ Line 571: / Line 569: @@
 	         <li><b>Purification vector</b></li>
 	         <ul>
-		    <li>This week we tried to amplify the T7_RBS promotor for the purification vector.</li>
+		    <li>This week we tried to amplify the T7_RBS promoter for the purification vector. Additionally we amplified the intein tag containing a chitin binding domain.</li>
                      <ul>
-                        <font color="red"><li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer1" target="_blank">Primer1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)</li>
+                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#RBS_int_rev" target="_blank">RBS_int_rev</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_int_fw" target="_blank">pSB1C3_int_fw</a>) of the promoter (T7_RBS)</li>
 	               <ul>
-		          <li>Annealing temperature: ...</li>
+		          <li>Annealing temperature: 55°C</li>
-		          <li>Bands (not) as expected (... bp)</li>
+		          <li>Bands as expected (~2000 bp)</li>
-	               </ul></font>
+	               </ul>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#int_RBS_fw" target="_blank">int_RBS_fw</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#int_pSB1C3_rev" target="_blank">int_pSB1C3_rev</a>) the intein tag with chitin binding domain (<i>intCBD</i>)</li>
+	               <ul>
+		          <li>Annealing temperature: 55°C</li>
+		          <li>Bands as expected (~1000 bp)</li>
+	               </ul>
+                       <li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li>
+                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with T7_RBS and <i>intCBD</i></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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
+            </li>
+	<ul>
+		<li>Annealing temperature: 55°C</li>
+		<li>Bands as expected (~3300 bp)</li>
+	</ul>
+              <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_T7_RBS_intCBD</li>
                      </ul>
 	         </ul>
@@ Line 587: / Line 601: @@
                      <li>As the purification of the carboxysome showed no results, we decided to induce protein expression with higher concentrations of IPTG.  </li>
                      <ul>
-<li> Cultivation was carried out using a modified method of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation%20for%20Expression%20of%20recombinant%20proteins" target="_blank">Cultivation for Expression of recombinant proteins </a>. Cells were continous cultivated by a temperature of 20 °C, and protein expressing was induced when the culture reaches an OD<sub>600</sub> of 0.9 - 1.2. IPTG in final concentrations of 0.5 mM, 2 mM and 5 mM was used.
+<li> Cultivation was carried out using a modified method of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation%20for%20Expression%20of%20recombinant%20proteins" target="_blank">Cultivation for Expression of recombinant proteins </a>. Cells were continous cultivated by a temperature of 20 °C, and protein expression was induced when the culture reaches an OD<sub>600</sub> of 0.9 - 1.2. IPTG in different final concentrations of 0.5 mM, 2 mM and 5 mM was used to verify the expression of the carboxysomal proteins through <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS-PAGE </a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Matrix-assistedLaserDesorption/Ionization%E2%80%93Timeofflight%20%28MALDI-TOF%29" target="_blank">MALDI-TOF </a> . Samples were generated using the protocol for <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">Fast Cell Lysis for SDS-PAGE. </a>
+<center>
-                    </ul>
+<table style="background-color:transparent">
+ <tr>
-                 </ul>
+  <td>
+<div class="element" style="margin:10px; padding:10px; text-align:center; width:220px">
+      <a href="https://static.igem.org/mediawiki/2014/e/ec/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_0.5_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/ec/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_0.5_mM.jpg" width="220px"></a><br>
+<font size="1" style="text-align:center;">Proteinexpression of pHnCBcsoS1D <br>induced with 0.5 mM IPTG</font>
+</div>
+  </td>
+  <td>
+<div class="element" style="margin:10px; padding:10px; text-align:center; width:220px">
+      <a href="https://static.igem.org/mediawiki/2014/c/cf/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_2_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/cf/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_2_mM.jpg" width="220px"></a><br>
+<font size="1" style="text-align:center;">Proteinexpression of pHnCBcsoS1D <br>induced with 2 mM IPTG</font>
+</div>
+  </td>
+  <td>
+<div class="element" style="margin:10px; padding:10px; text-align:center; width:220px">
+      <a href="https://static.igem.org/mediawiki/2014/a/af/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_5_mM.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/af/Bielefeld-CeBiTec_14-10-16_pHnCBcsoS1D_5_mM.jpg" width="220px"></a><br>
+<font size="1" style="text-align:center;">Proteinexpression of pHnCBcsoS1D <br>induced with 5 mM IPTG</font>
+</div>
+  </td>
+ </tr>
+</table>
+</center>
@@ Line 733: / Line 768: @@
 	<ul>
 		<li>Annealing temperature: 55 °C</li>
-		<li>Bands as expected (~900 bp)</li>
+		<li>Bands as expected (~600 bp)</li>
+                <div class="element" style="height:350px; width:120px; text-align:center">
+                      <a href="https://static.igem.org/mediawiki/2014/2/27/Bielefeld_CeBiTec_2014-10-17_T7_sRNA_pfkA_cPCR_08_27.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/2/27/Bielefeld_CeBiTec_2014-10-17_T7_sRNA_pfkA_cPCR_08_27.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. As a Ladder we used <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 from Thermo Scientific</a>. </font>
+                    </div>
 	</ul>
 <ul>
@@ Line 781: / Line 820: @@
                   </ul>
                </ul>
+<br>
+<li><b><i>Purification of the carboxysome</b></i></li>
+                 <ul>
+                    <li>The results of our experiments suggest that higher IPTG concentrations are needed for a efffective protein expression of the carboxysome. For this reason, we induced the protein expression with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal </a>, but slightly modificated. The culture volumen was upscaled to 1 litre. For a more effective cell lysis, the sonification protocol was modified using eight cylces a one min with one min cooling intervals for sonification. </li>
+&rarr; The purification was not succesful, as you could not recognize a visible band in the gradient.
+                    </ul>
+                 </ul>
 </ul>