Team:Bielefeld-Germany/Labjournal/September
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**Due to the illegal restriction site EcoR1, we repeated the digestion of rib and rib-nus only with XbaI | **Due to the illegal restriction site EcoR1, we repeated the digestion of rib and rib-nus only with XbaI | ||
- | [[Image:iGEM_Bielefeld_2013 | + | [[Image:iGEM_Bielefeld_2013._restriktion_von_rib_und_rib_nus_mit_Xba1._4.9.13.jpg|300px|thumb|center|<p align="justify"> '''Figure X: Test-digest of the plasmidisolations with EcoR1 and Spe1 from NEB. '''</p>]] |
**After the succesfull integration of the three most important sequences in pSB1C3, and the insofar completion of our first Biobrick BBa_K7211XXX (ribC), we started with the elimination of the illegal restriction sites in rib and norM. | **After the succesfull integration of the three most important sequences in pSB1C3, and the insofar completion of our first Biobrick BBa_K7211XXX (ribC), we started with the elimination of the illegal restriction sites in rib and norM. |
Revision as of 13:51, 2 October 2013
September
Milestones
Week 18
Organization
- This week, we supported the CeBiTec student academy with supervising the ‘Synthetic Biology’ experiment. In addition we had a presentation about iGEM and the Bielefeld iGEM projects.
- Our team assigned the Track Food & Energy in this year, with the project title ‘Ecolectricity – currently available’. Project abstract, safety forms and team roster were added.
MFC
Mediators
- Glycerol dehydrogenase
- Repeating NADH-Assay with three times PBS buffer washing step shows good results with better NADH production for E. coli KRX with pSB1C3 <bbpart>BBa_K1172203</bbpart> and <bbpart>BBa_K1172204</bbpart> compared with E. coli KRX wildtyp. GldA with T7 promotor (<bbpart>BBa_K1172203</bbpart>) shows 0,5 μM NADH overproduction and GldA with Lac promotor (<bbpart>BBa_K1172204</bbpart>) shows 0,3 μM NADH overproduction. The data refer to a culture with OD600 = 3.0.
- Second successful optimized NADH-Assay with Escherichia coli KRX Wildtyp strain and E. coli KRX with pSB1C3 and <bbpart>BBa_K1172203</bbpart>, <bbpart>BBa_K1172204</bbpart>, and <bbpart>BBa_K1172205</bbpart>.
- GldA with T7 promotor (<bbpart>BBa_K1172203</bbpart>) shows 1,7 μM NADH overproduction and GldA with Lac promotor (<bbpart>BBa_K1172204</bbpart>) shows 0,2 μM NADH overproduction. The GldA expression by the Anderson promotor (<bbpart>BBa_K1172205</bbpart>) is too weak for an efficient NADH overproduction. The data refer to a culture with OD600 = 5.0.
- Overexpressed glycerol dehydrogenase (Figure. 4 Week 17) should be examined by MALDI-TOF MS/MS.
- Tryptic digest of gel lanes for analysis with MALDI-TOF.
- Glycerol dehydrogenase was examined by MALDI-TOF MS/MS with a Mascot Score of 266 against Escherichia coli database.
- Overexpressed glycerol dehydrogenase (Figure. 4 Week 17) should be examined by MALDI-TOF MS/MS.
- Riboflavin
- We tried to ligate our four DNA-Sequences in pSB1C3 using Gibson-Assembly.
- To further minimize the risk of false positive clones just carrying pSB1C3, we digested the pSB1C3-PCR-products with DpnI, prior to Gibson.
- We assembled every fragment with its specific vector six times. For each fragment, we used two different approaches: The “Standard-protocol” where you take 100ng insert in 3:1 molar excess compared to the vector and an “over the top-protocol” (OTTP). The OTTP uses the same molar excess, but a much higher amount of DNA.
- After picking colonies on ¼ plates we processed the plasmidisolation and test-digested the samples to confirm that the DNA-sequences, still with the illegal sites, were integrated in pSB1C3. Digestions were performed with 250 ng of plasmid-DNA + 2,5 µl of Cut-Smart-Buffer (NEB) + 0,5 µl of enzyme each.
- Due to the illegal restriction site EcoR1, we repeated the digestion of rib and rib-nus only with XbaI
- After the succesfull integration of the three most important sequences in pSB1C3, and the insofar completion of our first Biobrick BBa_K7211XXX (ribC), we started with the elimination of the illegal restriction sites in rib and norM.
- Therefore we diluted one rib and one norM sample to 10 ng/µl and used it as PCR template.
- At the beginning of the week, we had started to design primers for this purpose:
- pst1-234 fwd (38 bp) TGTCACCTTAGAACCCTGTAGCCATTATGGTCGTACGC
- pst1-234 rev (32 bp) CGACCATAATGGCTACAGGGTTCTAAGGTGAC
- pst1-1644 fwd (30 bp) GTGCCCCATACTGTAGGTGAAACCACGTTG
- pst1-1644 rev (34 bp) AACGTGGTTTCACCTACAGTATGGGGCACAATCG
- EcoR1 fwd (43 bp) GGCACTCAGTTCACTTAGGTATAGAATTTATAACAACAGTCAC
- EcoR1 rev (43 bp) GTGACTGTTGTTATAAATTCTATACCTAAGTGAACTGAGTGCC
- pst1 norM fwd (38 bp) GCGATCGTGCTGATATTTCTGCGGTGGTCGCCAAAGTC
- pst1 norM rev (41 bp) CAATAAGCCGACTTTGGCGACCACCGCAGAAATATCAGCAC
Cytochromes
- New primers were ordered. pSB1C3 Gibson primer for the ccmAH cluster and a new reverse pSB1C3 primer for the mtrCAB cluster, which has a larger overlap and should therefore improve assembly efficiency.
- psB1C3_rev_ccm....(40nt) CTGGCTTCAAGCATACCCACCTAGAAGCGGCCGCGAATTC
- psB1C3_fwd_ccm....(41nt) CGCCGCAGGAGAGTAAATAATACTAGTAGCGGCCGCTGCAG
- psB1C3_rev_mtrCAB.(40nt) GATTTTTGTGCGTTCATCATCTAGAAGCGGCCGCGAATTC
- Repeated Amplification of psB1C3 with these new primers with corresponding Gibson-overlaps for mtrCAB and ccmAH.
- Phusion PCR
- Template: Plasmid pSB1C3::RFP (J04500) [5ng]
- Annealing: 60°C
- Elongation: 45s
- The PCR reaction was subsequently digested with DpnI to eliminate any remaining plasmid DNA.
- pSB1C3......4-0609-301......110ng/ul
- Repeated Amplification of fragment3 of the mtrCAB cluster.
- Frag3_fwd (30nt) :GTCTGCAATTATGGCATTAGTCGTCACACC
- mtrB_rev (41nt) :CTGCAGCGGCCGCTACTAGTATTAGAGTTTGTAACTCATGC
- Phusion PCR
- Template: S.oneidensis genomic DNA [5ng]
- Annealing: Gradient [60-70°C]
- Elongation: 50s
- A Gradient was applied to figure out the annealing temperature with the minimal false bands, so that a PCR clean up with higher yields instead of a gel extraction could be used for isolation. As Figure 4 shows the optimal annealing temperature is around 62-66°C.
- PCR clean up
- Fragment3.......4-0509-305......285ng/ul
- Repeated Amplification of the ccmAH cluster with Gibson primers.
- ccm_fwd (40nt) :GAATTCGCGGCCGCTTCTAGGTGGGTATGCTTGAAGCCAG
- ccm_rev (41nt) :CTGCAGCGGCCGCTACTAGTATTATTTACTCTCCTGCGGCG
- Phusion PCR
- Template: E.coli genomic DNA [5ng]
- Annealing: 68°C
- Elongation: 1:35
- The PCR was cleaned up and lyophilisated with the SpeedVac
- ccmAH......4-0509-301......157.0ng/ul
Biosafety
- We executed the characterization of araC by cultivation (200rpm) in shaking flasks with different carbon sources. We used the M9 minimal medium with glucose/arabinose/glycin to measure the growth and the fluorescence of our biosafety system. The fluorescence is caused by the protein GFP which is a replacement for Barnase which is applied for the characterization to draw conclusions from the theoretical expression of the RNase. In continious intervals samples were taken the growth of the cells were measured by absorbance and the fluorescence by a plate reader.
Porines
- Successful Hexadecan-Assay for characterization of hydrophobicity of the outer membrane. Using Escherichia coli KRX Wildtyp and comparing it with:
- <bbpart>BBa_K1172502</bbpart>: <bbpart>BBa_K1172501</bbpart> + <bbpart>BBa_K525998</bbpart>
- <bbpart>BBa_K1172503</bbpart>: <bbpart>BBa_K1172501</bbpart> + <bbpart>BBa_J04500</bbpart>
- <bbpart>BBa_K1172504</bbpart>: <bbpart>BBa_K1172501</bbpart> + <bbpart>BBa_K608007</bbpart>
- <bbpart>BBa_K1172505</bbpart>: <bbpart>BBa_K1172501</bbpart> + <bbpart>BBa_K608006</bbpart>
- <bbpart>BBa_K1172507</bbpart>: <bbpart>BBa_K1172501</bbpart> + <bbpart>BBa_K608002</bbpart>
- The hydrophobicity increases continuously with increasing promoter strength up to 221% hydrophobizity in contrast to wildtyp strain.
- Successful ONPG and NPN uptake assays were performed. Membrane permeability is continuously increasing from weak to strong promoter strength.
- Beside testing OprF Biobrick (<bbpart>BBa_K1172501</bbpart>) with different assays and SDS-PAGE, the membrane should be visually displayed. The technique of choice is atomic force microscopy.
- The glass slides were coated with Escherichia coli KRX wild type and Escherichia coli KRX with <bbpart>BBa_K1172502</bbpart> in order to compare the membrane differences. The AFM itself was performed with the help of the working group of Prof. Dr. Dario Anselmetti, with special help from Dr. Volker Walhorn.
Week 19
Organization
- Our WDR TV contribution could be seen in the ‘OWL local time’ and in the WDR library.
- We have successfully performed the day of synthetic biology in the city of Bielefeld. We were able to explain many people our project with the help of our poster and the kids had a lot of fun with performing different experiments for introducing them into scientific work. DNA isolation from fruit and vegetables, pipetting of bright colors, chromatography with markers, a potato battery and microscopy were on the program. All in all a very successful day.
MFC
Mediators
Cytochromes
- Attempted another Gibson assembly with psB1C3 and the three mtrCAB fragments. The inserts were added equimolar and in a 3-fold excess to the vector.
psB1C3 | 4-0609-301 | 110ng/ul | 0.6ul | 66ng |
Fragment1 | 4-2608-302 | 48.8ng/ul | 3.1ul | 151ng |
Fragment2 | 4-2008-304 | 68.3ng/ul | 0.4ul | 27ng |
Fragment3 | 4-0509-301 | 285ng/ul | 0.9ul | 256ng |
- Various cells of the transformation were transfered to new plates and incubated again.
- Subsequently plasmids were isolated from this cultures and a restriction analysis with the enzymes EcoRI and PstI and a total amount of 500ng isolated plasmid DNA was performed.
- The resulting fragment should be the respective insert mtrCAB with a size of approx. 5.1kb. The Results are shown in Figure 1 below. The correct sample is located in lane 12.
- An aliquot of the plasmid DNA was used for sequencing.
- The corresponding results verify the correct insertion of the mtrCAB cluster into the psB1C3 backbone.
- Another Gibson assembly was attempted with the ccmAH cluster. The insert was added in a 3-old excess to the vector.
4-0609-303 | 4-0509-301 | 75.7ng/ul | 0.7ul | 52.5ng |
ccmAH | 4-0509-301 | 157.0ng/ul | 3.1ul | 486.7ng |
Part | Vector | Insert | Volume |
---|---|---|---|
K608006::mtrCAB | 50ng | 374ng | 20ul |
K608002::mtrCAB | 50ng | 374ng | 20ul |
K525998::mtrCAB | 50ng | 374ng | 20ul |
K608006::mtrCAB | 10ng | 150ng | 10ul |
K608002::mtrCAB | 10ng | 150ng | 10ul |
K525998::mtrCAB | 10ng | 150ng | 10ul |
- Notes: Interestingly the ligations with only 10ng vector and 6-fold excess of the insert seemed to be more effecient, since they yield significantly more colonies.
- The succesfull ligation was confirmed via restriction analysis. The isolated plasmids from the aforementioned transformations were digested with the enzyme EcoRI, which should linearised them and generate a 7.2kb fragment. The Results are shown in Figure3 and verify the correct ligation, leading to the following succesfully constructed devices:
- <bbpart>BBa_K1172403</bbpart>: mtrCAB cluster with Anderson 0.33 and weak rbs
- <bbpart>BBa_K1172404</bbpart>: mtrCAB cluster with Anderson 0.77 and strong rbs
- <bbpart>BBa_K1172405</bbpart>: mtrCAB cluster with T7 and strong rbs
- Subsequently glycerol stocks were made from the ligations and the further characterisation was started.
Biosafety
- We transformed the tetracyclin repressor into KRX via electroporation for plasmid isolation. After this we isolated the plasmids and other plasmids which are listend below.
- BBa_C0040_3 250,2 ng/µL (9-108-451)
- BBa_C0040_4 124,5 ng/µL (9-108-452)
- Ptac_alr1 115,8 ng/µL (9-108-453)
- Ptac_alr5 68,1 ng/µL (9-108-454)
- Ptac_alr6 163,6 ng/µL (9-108-455)
- DNase Ba3 86,6 ng/µL (9-108-456)
- We did purification of the restriction and measured the dna concentration via nanodrop:
- BBa_C0040 TetO: 35,0 ng/µL
- B0015 Terminator 22,1 ng/µL
- GFP 4,4 ng/µL
- Alr 8,7 ng/µL
- After we isolated the different plasmids we assembled the alanine racemase with the terminator (<bbpart>BBa_K1172003</bbpart>), TetO with GFP (<bbpart>BBa_K1172014</bbpart>) lacI+terminator. We transformed them into competent cells and did plasmid isolation.
- LacI (C0012) 86,5 ng/µL (9-129-451)
- Terminator (B0015) 95,4 ng/µL (9-129-452)
- Terminator (B0015) 46,1 ng/µL (9-129-453)
- Alr_Terminator 84,6 ng/µL (9-129-454)
- Alr_Terminator 65,5 ng/µL (9-129-455)
- Alr_Terminator 33,4 ng/µL (9-129-456)
- Alr_Terminator 98,2 ng/µL (9-129-457)
- TetO_GFP 102,6 ng/µL (9-129-458)
- TetO_GFP 77,1 ng/µL (9-129-459)
- TetO_GFP 91,8 ng/µL (9-129-460)
- After the plasmid isolation we checked with a restriction analysis with EcoR1 and Pst1 if the correct inserts are in the vectors. The results are schown below.
- We send them for sequence analysis to the sequencing service. The results confirmed our constructs.
- We did a PCR to get araC. It was done by executing the phusion protocoll and recipe. The results are listened below:
- After cheking the PCR result we purified the samples with the PCR clean up Kit and measured the DNA concentration.:
- araC 12a 58,8 ng/µL (9-129-463)
- araC 12b 84,1 ng/µL (9-129-464)
- We did PCR with Tetracylin repressor purificated the samples and did gelelectrophoresis:
- We assembled the three front constructs of our biosafety system via Gibson Assembly:
- BSVaraC
- 1,5 µL pRha (BBa_K914003)
- 2,3 µL alr_terminator (BBa_K1172003)
- 1,5 µL araC
- BSVTetR
- 1,5 µL pRha (BBa_K914003)
- 2,3 µL alr_terminator (BBa_K1172003)
- 1,5 µL TetR (BBa_C0040)
- BSVlacI
- 1,0 µL pRha (BBa_K914003)
- 7,0 µL lacI (BBa_C0012)
- 0,7 µL alr_terminator
- We had assembled TetO with GFP before. pBAD with GFP and plac with GFP were already in the parts registry.
- Back constructs:
- BSVaraC
- pBAD+GFP
- BSVaraC
- BSVTetR
- TetO+GFP
- BSVTetR
- BSVlacI
- plac+GFP
- BSVlacI
Porines
Week 21
Organization
MFC
Mediators
- Glycerol dehydrogenase
- Glycerol dependent NADH-Assay with Escherichia coli KRX Wildtyp strain and E. coli KRX with pSB1C3 and <bbpart>BBa_K1172203</bbpart> by testing different glycerol concentrations of M9-medium.
- NADH-Assay shows an increasing intra- and extracellular NADH concentration for <bbpart>BBa_K1172203</bbpart> with increasing glycerol concentration in M9-medium.
Cytochromes
- For characterisation the mtrCAB cluster in with the three different promoters <bbpart>BBa_K1172403</bbpart>, <bbpart>BBa_K1172404</bbpart> and <bbpart>BBa_K1172405</bbpart>,the latter induced and uninduced were cultivated anaerob to ensure the the expression of the ccmAH cluster.
- Subsequently the periplasmic and membrane protein fraction from E. coli was released by cold osmotic shock
- Samples from each fraction were analyzed by SDS-PAGE.
- The resulting gel did not show a significant difference between the samples and the control
- Furthermore a cytochrome type c redox activity assay for the different fractionations was carried out.
- The absorbance was measured at wavelength between 350 and 650nm.
- The oxidized cytochromes were expected to have peaks at 410nm and 530nm.
- The 410nm peak could be measured around 400nm
- The reduced ones should show a shift to 420nm and 552 nm, respectively.
- No significant peak could be measured in this area.
Biosafety
- We cultivated the different biosafety systems for measuring how the specific product building rate looks like in M9 mediumwith different carbon sources. As mentioned before, we used GFP instead of Barnase for characterisation. We sampled in regulary intervals to measure the absorbance and the fluorescence.
Porines
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