Team:Slovenia/Notebook/July.html

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July 1st, 2009
BL21DE3 pLysS cells transformed with vectors, containing d53 and Dimtetra, were inoculated into 100 ml LB media. All remaining PCR products (from June 29th) were isolated from gel.

July 3rd, 2009
We prepared competent cells BL21DE3pLysS. These cells will be used for production of proteins. We prepared primers (see June 29th).

July 6th, 2009
PCR for amplification of certain sequences from previously made plasmids to make the following BioBricks: ccdB, gyrB, LL-37, split YFP, CutA1, foldon domain. We used a single E. coli colony to amplify CutA1. For the amplification of foldon domain only primers were used (partially complementary). We made apropriate primer and template dilutions for any further PCRs with these primers. CutA1 amplified from a single E. coli colony. We made a 15% gel for SDS PAGE to confirm the presence of d53 and dimtetra in cell lysate and inclusion bodies. We ran an SDS-PAGE electrophoresis. Also: - samples for Western blot were prepared - 100 ml of LB with ampicillin were inoculated with Amp resistant dimtetra2 producing E. coli BL21 and put on a shaker until the following morning. - samples were sent for sequencing to confirm the identity of inserts

July 7th, 2009
Dimtetra2 producing overnight culture of Amp resistant BL21 was inoculated into 4 flasks of LB (with ampicillin) - a 2L fermentation. We measured OD from time to time until bacteria grew to OD from 0.6 to 0.8. Then we induced production of dimtetra2 with IPTG. Production lasted for 4 hours and 45 minutes. Broth was centrifuged and the resulting pellets were saved. Yesterdays PCR reactions were put on agarose electrophoresis (2,2% & 1,2%, depending on the molecular weight of the product). Results were positive. When we photographed agarose gels we also took photos of yesterdays SDS PAGE. Afterwards, the PCR amplified fragments were purified. Also: - 3 new PCR reactions to amplify KSI-DP, p53 and a part of a polilinker - inoculation of 100 ml of LB with Amp with d53 producing E. coli - preparation for tomorrows fermentation.

July 8th, 2009
We used a d53 producing overnight culture of BL21 strain to set up another 2L fermentation, this time to - obviously - produce d53. When apropriate OD was reached, we induced production of d53 with IPTG. Production lasted for 5 hours and 20 minutes. Broth was then centrifuged and the resulting pellets were frozen. Meanwhile we tried to isolate dimtetra2 from yesterdays fermentation. We used a lysis buffer to resuspend and lyse pellets of cell material and added CPI - a protease inhibitor. After a bit of waiting we sonicated the cells, centrifugated and after that we collected the supernatant and saved it. Because of SDS PAGE we assumed dimtetra2 was located in the inclusion bodies (IB) so we washed the IB with lysis buffer and centrifugated again. We saved a majority of IB on -80°C and we used 1 tube to resuspend IB in MQ and dissolve in guanidinium hydrochloride. With these samples we did a practice study with circular dichroism spectrometer. We also did an agarose electrophoresis of PCR products, which showed that the PCR failed. Therefore, we tried again, this time with new dNTPs. Transformation of DH5a with our newly arrived construct (PL H1 GCN4 p1) was also performed and the cells were put on a plate with kanamycin. All in all it was a busy day, one of many more to come ...

July 9th, 2009
We used pellets from yesterdays fermentation to isolate d53. We used the same procedure like we used to isolate dimtetra2 ... except we used the supernatant after centrifugation because d53 is soluble. We measured protein concentration with BioRad protein assay. After we prepared many buffers for Ni-NTA column chromatography and peptide SDS-PAGE, we prepared Ni-NTA column. Supernatant with d53 was poured on the column and shaked over night. Dimtetra samples were dialysed over night in MQ and Ni2+ solution. PCR worked. Remainder of PCR reaction was put on the gel and products were cut out and isolated from it. Restriction of pSB1A2 (M13) plasmid with PstI and EcoRI. Transformation: •	pSBA2 (M13) into DH5α cells. •	In order to test BL21 cells (previously made by Nika and Marko) for competence, we transformed them with pLuc plasmid. PCR ligation for creating a polilinker site was also prepared.

July 10th, 2009
Samples of dimtetra2 were taken out of dialysis tubes. We carefully washed the tubes with MQ and saved in 20% ethanol in a refrigerated falcon tube. We noticed a precipitate after dialysis in Ni2+ solution. We continued and finished the isolation of d53 on Ni-NTA column. Elution fractions were put to dialysis overnight. We also did SDS PAGE, which showed nice results. After that we got to know the fluorimeter and we did another analysis with CD - this time it showed the presence of an alpha structure in refolded dimtetra2. We ran the PCR reaction that was prepared yesterday (PCR ligation). Product was put on a gel, which revealed that reaction was not successful. Therefore, another PCR ligation was set under different conditions. PCR products were later frozen to be analysed on Monday. Restriction products of plasmid pSB1A2 (July 9th) were also put on a gel and then isolated. Plasmid containing PL_H1_GCN4_p1 construct was isolated from 10 mL culture (transformation: July 8th). Plasmid was then cut with HindIII and XhoI.

July 13th, 2009
Previously diluted and frozen dimtetra2 samples were taken to CD analysis (lambda=(190, 260)nm) which showed certain helical structure. We also did thermal stability scan at few chosen wavelenghts and found one melting temperature - measured melting temperature = 59,8oC +/- 1,2oC (using Boltzmann fit) Products from Friday's restriction reaction were put on a gel. Then 2 fragments were isolated from gel: •	a fragment we will need to prepare vector without His-tag •	remaining part of the plasmid Isolated fragments were further cut with EcoRI and XbaI (fragment for vector without His-tag) and EcoRI and PstI (remaining plasmid). Products of PCR ligation were also put on a gel, but they were not sufficient for isolation. So, we ran yet another PCR ligation with different DNA polimerase testing different conditions.

July 14th, 2009
This time results of PCR ligation were successful and fragment was isolated from gel. The fragment was then cut with EcoRI and PstI. Product of restriction of part of plasmid (July 13th) was put on a gel. Restriction of the remaining plasmid was not successful, so we inoculated DH5a cells with PL_H1_GCN4_p1 plasmid for the miniprep again. Fragment for vector without His-tag was cleaned with Nucleotide removal kit. pSB1A2 vector was ligated with the ccdB domain to prepair the vector with His-Tag at the C-terminal end of the construct. We took photos of peptide SDS PAGE. We will repeat it because proteins did not separate as good as we thought they should. Next time we will use less protein. Precipitate of d53 which appeared after dialysis in MQ was collected and frozen (-80°C). The soluble part was concentrated, dissolved in 50 mM Tris, pH=8,0 and then we measured absorbance at 280 nm. Then we tried measuring after adding 150 mM NaCl – absorbance was a bit lower. A part of precipitate was dissolved in 6M GvHCl (50 mM Tris, pH=7,0) and then dialysed in 50 mM Tris, pH 8,0. IBs of dimtetra were also dissolved in 6M GvHCl (50 mM Tris, pH=7,0) but dialysed in MQ. After we made the buffer for gel filtration (50 mM Tris, pH=8,0), we prepared the column for tomorrows filtration. Another CD - this time with d53 (soluble). Alpha helix pattern was seen and melting temperature was calculated: 80,7 +/- 3,4oC.

July 15th, 2009
PL_H1_GCN4_p1 plasmid was isolated and cut with EcoRI in PstI. Fragments were put on gel and 128bp long fragment was isolated from the gel. This fragment was then ligated into the pSB1A2 vector to prepare vector with His-tag at the N-terminal of the construct. Many different buffers for dissolving d53 and dimtetra were tested. d53 precipitates in 50 mM Tris (pH=8). Dialysis of d53 and dimtetra in 20 mM Na3PO4 and 150 mM NaCl was set up. d53 was put on gel filtration. The standard we used was chymotrypsinogen. Results showed that d53 is in a dimer form.

July 16th, 2009
The dimtetra2 and d53 samples were inspected with TEM. All in all not much has been seen, because they were both too dense (c(dimtetra)=3,75 mikrog/mikrol, c(d53)=0,22mikrog/mikrol), though there were a few photos showing ordered structures and strand-like patterns( photos uploaded on GoogleGroups). Transformation of DH5a cells with newly arrived vectors containing constructs P1,P2,P3,P4 (in one vector) and P5,P6,P7,P8 (in another vector). We concentrated dimtetra and d53 after dialysis in 20 mM Na3PO4 and 150 mM NaCl. Concentration of dimtetra was too low to be used for gel filtration but d53 was OK. A standard borrowed from Jernej and lysozyme were used.

July 17th, 2009
Transformation of: -DH5a cells with vector with His-tag at the N-terminal of the construct -DB3.1 cells with vector with His-Tag at the C-terminal of the construct Colonies of cells, transformed with vectors containing constructs P1,P2,P3,P4 and P5,P6,P7,P8, from the plates were inoculated for miniprep. We tried mixing dimtetra and d53 in different molar proportions (3:1, 1:1 and 1:3). We started with proteins in 6M GvHCl (+ 50 mM Tris, pH=7) and then used two different methods – squirting cca 250 l of the mixtures into 10 ml beakers with MQ and a magnetic stirrer (quick method) or slowly injecting MQ while vortexing an epruvete (slow method). We saw dust-like particles in all cases, although we did measure A280 after concentrating these mixtures and found that most soluble proteins remained in the mixture of dimtetra and d53 in 1:3 proportions. Equimolar mixture has shown far less soluble proteins using the slow method than was shown with the quick method. 3:1 showed little soluble protein.

July 21st, 2009
Vectors with His-tag at the N-terminal of the construct and His-Tag at the C-terminal of the construct were isolated. We performed restriction analysis to confirm the presence of appropriate polilinker in vectors. The polilinker with His-tag at the N-terminal of the construct was successfully ligated into the vector, but the polilinker with His-tag at the C-terminal of the construct was not. Vector with His-tag at the N-terminal of the construct was isolated from colonies and stored for further usage.Vector with His-tag at the N-terminal of the construct was isolated from colonies and stored for further usage. CD analysis: 10uM dimtetra was scanned in the far (190-230nm) UV spectrum and between 300 and 800nm (we used 1mm cell). We gradually added Ni to protein samples and observed the secondary structure. We added 2uM, 10uM and 20uM Ni, but the helical structure remained folded. The spectrum from 300-800 nm was a bit more problematic, because the HT was too high (the result of too high absorbance). We should have used 1 cm cell and c=1mg/mL concentration (similarly: 1 mm cell and c=0,1mg/mL for the far). Peptide SDS was done. Absorbance of dimtetra in bis-tris was measured. It doesn't look promising. Cross-linking was done with two different reagents (BS3 and sulfo-SANPAH) and SDS PAGE samples were prepared.

July 22nd, 2009
We cut the vector with His-tag at the N-terminus of the construct and ccdB domain with the same enzymes and then ligated domain into vector. We set PCR ligation of ccdB domain and polilinker. Product will be ligated in psb1A2 vector resulting in vector with His-tag at the C-terminus of the construct. CD in the 'near' spectrum was done again. We added measurements for the far sp. for d53 sample from yesterday (10uM) and then prepared new samples with higher concentration (dimtetra: 2,25mg/mL and d53: 0,65mg/mL). D53 was mixed with 50uL MQ (to get enough volume) and analysed - there were no strict signs of Ni binding. It is not certain whether this is because there wasn't one or because we didn't have proper signal. We will try to do the measurment again tomorrow with a protein that certainly binds Ni and then analyse dimtetra again. We made restriction of PCR products as follows: for restriction of KSI-DP we used EcoRI/Pst; for restriction of gyrB, LL-37, nYFP (PCR5+6), cYFP, CutA1, foldon, p53, P1, P2, P3, P4, P5, P6, P7 and P8 we used NgoMIV/SpeI. We purified restricted fragments using QIAquick Nucleotide Removal Kit and measured their concentrations. Many different solvents were tested for refolding of dimtetra – MOPS, PIPES, HEPES, ... which failed. Mixtures with acetonitrile showed positive results – 20% acetonitrile 50 mM sodium acetate pH 5,2. We prepared -         6 M GvHCl pH 4,5 -         20% acetonitrile 50 mM sodium acetate pH 4,5 and dissolved some d53 precipitate in 6 M GvHCl pH4,5.

July 23rd, 2009
We prepared 50 flasks with 10mL of LB. Products of PCR ligation (of ccdB domain and polilinker) were separated on electrophoresis gel and then isolated. They were cut with appropriate enzymes (EcoRI/PstI) and ligated in psb1A2 vector resulting in vector with His-tag at the C-terminus of the construct. DB3.1 cells were transformed with: -vector with His-tag at the C-terminus of the construct -vector with His-tag at the N-terminus of the construct, containing ccdB domain -backligation of pSB1A2 vector -backligation of vector with His-tag at the N-terminus of the construct, without ccdB domain We did another refolding experiment with different proportions of dimtetra and d53 using the quick method as on Friday (July 17th). We used dissolved proteins in 6 M GvHCl pH 4,5, mixed them in 3:1, 1:1 and 1:3 proportions and refolded them in 10 ml of 20% acetonitrile/50 mM sodium acetate/pH 4,5. dimtetra was dissolved in 6 M GvHCl pH 4,5 & 50 mM NaAc and refolded in 20% acetonitrile/50 mM NaAc/pH 4,5. Refolding mixtures were then concentrated and A280 measured. Results suggest that all of the protein precipitated.

July 24th, 2009
We multiplied polilinker and ccdB domain from colonies, that had been transformed on 23rd July, directly using Colony PCR to confirm the presence of appropriate inserts in vectors (one with His-tag at the N-terminus of the construct and one with His-tag at the C-terminus of the construct). SDS PAGE was done. A photo will be taken on Monday.

July 27th, 2009
Several colonies containing vectors with His-tag at the N-terminus of the construct and with His-tag at the C-terminus of the construct were inoculated into 10 mL of LBAmp. We multiplied KSI-DP-P2-33 (PCR) and isolated it from gel. We concluded CD analysis with the sample of d53 enriched with different concentrations of Ni. The spectrum between 300 and 800 nm was measured, this time using ~1mg/mL protein concentration, 10mm cell and 4 repeats for averaging. Refolding of dimtetra and d53 in 20% acetonitrile. Dialysis of d53 from 6M GvHCl to crosslinking buffer overnight.

July 28th, 2009
Vectors with His-tag at the N-terminus of the construct and with His-tag at the C-terminus of the construct were isolated. Isolated vectors were cut so that constructs could be ligated into them. Both DB3.1 and DH5α cells were transformed with isolated vector with His-tag at the N-terminus of the construct and vector with His-tag at the C-terminus of the construct. PCR product (KSI-DP-P2-33) was cut with appropriate enzymes. We prepared a gel for another peptide SDS PAGE. When refolded from 6M GvHCl pH 7,0 into 20% acetonitrile, d53 precipitates, whereas dimtetra stays quite soluble. Another refolding experiment was done - this time with dimtetra and d53 from 6M GvHCl pH 4,5 into 20% acetonitrile. Dialysis: - 2x550 l of refolded dimtetra in 20% acetonitrile. - 1mg of d53 precipitate in 400 l of 6M GvHCl pH7.0 into crosslinking buffer (20 mM NaH2PO4, 150 mM NaCl, pH8) - twice in 500ml of buffer.

July 29th, 2009
Cut vectors from yesterday were isolated from gel. Constructs H1, GCN4, P1, P2, P3, P4, P5, P6, P7 and P8 were ligated into vector with His-tag at the N-terminus of the construct separately. Ligation products were transformed into DH5α cells. Vector without His tag was prepared.Vector without His tag was prepared. Refolding: dimtetra:d53 - 1:2 in 20% acetonitrile. The resulting spectrum was strange, maybe agregates still formed. Dimtetra was stable in 20% acetonitrile after dialysis. We failed to dialyse 1mg of d53 precipitate from guanidine into crosslinking buffer. d53 precipitated completely. We stored the precipitate at -80 °C. Dimtetra and d53 (refolded yesterday - 6M GvHCl pH 4,5 -> 20% acetonitrile, concentrated and stored in refrigerator) spectra were measured - peptides stayed quite soluble.

July 30th, 2009
More of vector with His-tag at the N-terminus of the construct was cut so that constructs could be ligated into it. ccdB domain with different pattern of restriction sites and terminator which adds additional 4 E to C-terminus of the construct inserted in vector were multiplied using PCR. Afterwards they were isolated from gel. Two sets of primers were annealed thus forming genes for peptide for binding zinc oxide and peptide for binding gold nanoparticles. Colonies of transformed cells from yesterday were inoculated from plates to 10 mL of LBAmp. Elution of d53 from cell lysate (stored at -80°C) on NiNTA column to get more soluble d53 and less 14k protein (extra band on SDS PAGE). This time we washed the column a little longer with buffers with less imidazole in them. In the end we dialysed 30 ml of diluted elution fraction in 20% acetonitrile and 10 ml in crosslinking buffer (20 mM sodium phosphate, 150 mM NaCl, pH8). Spectrum of dimtetra (result of a refolding experiment, dialysed in 20% acetonitrile) was measured. Spectrum of d53 in 20% acetonitrile (result of yesterdays refolding experiment) was measured.

July 31st, 2009
We ligated Z1 and AUG constructs into vector with His-tag at the N-terminus of the construct and transformed both of them into DH5α and inoculated them on LBAmp plate. Restricitons: PCR product (MCS-P-ccdB) with NgoMIV/PstI; PCR product (4E) with BspEI/PstI; vector without His tag with BspEI/PstI. We transformed T1W-EEE plasmid into DH5α and inoculated them on Kan plate. Dialysis: d53 was put into fresh solutions (crosslinking buffer and 20% acetonitrile). 400 l of d53 in crosslinking buffer was taken out and dialysed in another 500 ml crosslinking buffer, so we could do crosslinking faster. Crosslinking was done with sulfo SANPAH reagent in the same molar ratio with freshly eluted d53 as previously in the first crosslinking experiment. (22,2 x molar excess). Then we did 15% SDS PAGE and peptide SDS PAGE. Gels were stained, washed and photographed.

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