Team:Slovenia/Notebook/August.html

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August 3rd, 2009
We isolated vector without His tag and vectors containing following constructs: H1, GCN4, P1, P2, P3, P4, P5, P6, P7 and P8 (all are in vector with His tag at the N-terminus of the construct). We performed control restriction which showed that vectors contain appropriate inserts. We prepared front vectors and front inserts for assembling biobricks consisting of two inserts. Transformation of T1W_EEE_T1W_AEA plasmid into DH5a --> inoculation on Kan plate. Restrictions: we prepared MCS-P-ccdB and 4E segment for ligation into vector with His-tag on the N-terminus of the construct. Dialysis: 300 l dimtetra (A=0,28 x 10; 6M GvHCl/50 mM TRIS/pH7.0 -> MQ) for crosslinking. Samples of dialysed d53 (in crosslinking buffer and 20% acetonitrile) were frozen at -80°C on Friday. Today we thawed them and measured their spectra. We also measured spectra of dialysed d53 in refrigerator. Spectra were comparable, although -80°C samples showed slightly higher absorbance. Absorbance was a little lower than on Friday.

August 4th, 2009
BL21DE3 pLysS cells were transformed with vectors containing following constructs: H1, GCN4, P1, P2, P3, P4, P5, P6, P7 and P8 (all are in vector with His tag at the N-terminus of the construct). 4 L of LB were prepared and autoclaved for fermentation and production of P1, P2, P3 and P4. 4 15% SDS PAGE gels and 2 peptide SDS PAGE gels were made. 10 mL flasks with LB with ampicillin were inoculated with colonies of transformed BL21 cells (P1, P2, P3, P4) - preparation for fermentation. P1 was transformed into borrowed BL21 cells for comparison. Crosslinking was done with BS3 reagent, but this time in MQ. Old soluble d53 (MQ) was used as a positive control - it would show if BS3 works in MQ or not. We used 1 mM BS3 for d53 and 1 mM, 2.5 mM and 5 mM for dimtetra. After crosslinking, we noticed a lot of precipitate in dimtetra samples. Then we prepared them for SDS PAGE and stored at -20°C.

August 5th, 2009
Colony PCR was performed to check if ligation of front vectors and front inserts was successful. It showed that it was. Colonies were inoculated for mini-preps. PCR products (GyrB, L37, cYFP, CutA1, foldon, p53 and nYFP) were ligated into previously cut vector with His tag at the N-terminus of the construct. Isolation of plasmids containing T1W_EEE_T1W_AEA, Z1 and AUP. Restriction of vector with His-tag on N-terminus of the construct and restriction of vector without His-tag. Crosslinking samples were put on SDS PAGE. We saw that d53 crosslinked, so BS3 works even in MQ alone. We saw blind dimtetra very clearly but the crosslinked samples were pale (as was d53). We believe we can see dimers, but higher multimers are too pale. Peptide standard did not separate well. We should put more of dimtetra on SDS PAGE (luckily, we still have enough of the samples), because most of it stayed in the pockets. 100 ml of LB were inoculated with 100 ul of culture. This was done for P1, P2, P3, P4. Cultures were incubated over night at 37 °C, 160 RPM. We started preparations for preparing the samples for TEM. - 15 ml of d53 (30.7. elution) in 20% acetonitrile was concentrated until concentration rose from 0.319 mg/ml to 1.49 mg/ml. - dimtetra in MQ was also used (2,1 mg/ml). Mixtures were prepared - 50 ul 40 uM d53 + 10.7 ul 13 uM dimtetra + 139.9 ul crosslinking buffer - 10.7 ul dimtetra + 50 ul 20% acetonitrile + 139.9 ul crosslinking buffer - 50 ul 40 d53 + 139.3 ul crosslinking buffer + 10.7 ul MQ Mixtures were incubated over night at room temperature.

August 6th, 2009
Biobricks containing two inserts were isolated and cut to prepare front vectors. Front inserts were ligated into front vectors resulting in biobrick containing 3 inserts and His-tag at the N-terminus of the construct. DH5α cells were transformed with vectors (with His tag at the N-terminus of the construct) containing PCR products. Isolation of vector with His-tag on N-terminus of the construct. Gel electrophoresis: not all vector restrictions from previous day were not successfull. Control restrictions of vectors with His-tag on N-term. with inserted Z1 and AUP. T1W_EEE and T1W_AEA wer isolated from gel separately. It seems as if dimtetra precipitated a lot in the TEM mixtures overnight according to the spectra. 100 ml cultures were measured, a part of inoculum was transferred into 1L of LB with ampicillin for final stage of fermentation so the new OD600 was 0.15. 100 ml of P4 were put away - they would not be induced by IPTG. Cells were induced when their OD600 was between 0.6 and 0.9 with final IPTG concentration being 1mM. We induced these cells at about 10.30 except for P3 which were growing a bit slow - they were induced later. Fermentation ended at 16.00 for P2 and P4, at 17.45 for P3. We left P1 to grow overnight, since they surprisingly grew very slowly after induction. When fermentation ended we centrifuged the broths for 10-15 min, 5000 RPM ("the big centrifuge"). P3 pellets were frozen at -80°C, whereas P2 and P4 were lysed. P2 and P4 were lysed with cca 25 ml D1 lysis buffer (6 M guanidine hydrochloride / 100 mM Na2HPO4 / 10 mM TRIS / pH 8.0), a part of P2 was also lysed with deoxycholate lysis buffer. CPI was also added. Lysates were then stored at -80 °C. Denaturing buffers for isolation on Ni-NTA column were prepared.

August 7th, 2009
P1 culture was centrifuged and stored at -80 °C. Lysates (P2 and P4) were sonicated and then centrifuged for 10 min at 10000 RPM. Supernatants were stored at -80°C. A part of P2 supernatant in DOC was prepared for peptide SDS PAGE. We did a blind run of Ni-NTA columns with the buffers we were going to use. Acetonitrile/TFA elution mixture was made.We did a blind run of Ni-NTA columns with the buffers we were going to use. Acetonitrile/TFA elution mixture was made. TEM samples were prepared. Grid is put on a 15 l droplet of sample for 3 minutes. After that, we washed the grid with 3 droplets of 15 l MQ, one droplet at a time. Then the grid is put on a droplet of uranil acetate for 1.5 min. Then we dry the grid and put it in a marked slot in the little box. Samples: - 110 l 0.1 mg/ml d53 in 20% acetonitrile - 110 l 0.1 mg/ml d53 + 0.1 mg/ml dimtetra in 20% acetonitrile - 100 l 20% acetonitrile + 10 l 1mg/ml dimtetra - dimtetra in MQ, 0.09 mg/ml - control: poly-L-lysine with nickel sulfate TEM analysis -Sample F2 (poly-L): First we inspected control sample to determine whether Lysine (used for binding d53 and dimtetra onto grid) could be responsible for the pattern on the previous TEM photos. We found line patterns on the outskirts of the sample (see F2-1 and F2-2). Lines resembled ordered crystalline structures, so we conducted RTG diffraction to be certain (see F2-3). Diffraction pattern clearly confirmed our hypothesis and EDS analysis (see F2-4) showed that we probably see Ni (or S or mixed) crystals. It should be pointed out that we may have caused generation of crystals by 'overheating' the sample (via electron beam), because it seemed that we saw line pattern only after a certain amount of time. After quite a while we also found fibrile-like structures (see F2-5 and F2-6). -Sample C3 (d53 in AcN) First we saw linear packing in all directions on scale 200nm (see C3-1) - maximum resolution showed bazillion crystalline structures, as seen on F2, although we haven't used poly-L in this sample (see C3-2 which is zoomed C3-1 and C3-3). Crystals were found on different hights (inspected via alternating focus) as if they were stocked one on top of the other. Polymorph crystalline structure was confirmed with RTG diffraction (see C3-4) and EDS (see C3-5) showed that crystals were mainly made out of U (sample was contrasted with uranyl acetate). DH5a competent cells were prepared. Vectors with His-tag at the N-terminus of the construct, His-tag at the C-terminus of the construct and without His-tag were sent for sequencing. Several biobricks containing 3 inserts were transformed in DH5a cells. Biobrick containing 2 inserts was transformed in BL21(DE3)pLysS cells for production of proteins.

August 9th, 2009
Lysate supernatants of P2 and P4 were loaded on Ni-NTA columns and shaken overnight.

August 10th, 2009
Vectors with His-tag at the N-terminus of the construct, containing GyrB, L37, cYFP, CutA1, foldon, p53 and nYFP were isolated using GeneJET kit. A ligation to obtain vector with His-tag at the N-terminus of the construct was performed once again, because control restriction of final vector with His-tag at the N-terminus showed that previous ligation wasn’t successful. Vectors with His-tag at the N-terminus of the construct, containing three consecutive inserts were isolated. T1W_EEE and T1W_AEA were restricted and ligated into vector with His-tag on N-term. of the construct. Control restrictions of Z1 and AUP (that had been inserted into vector with His-tag at the N-terminus) showed that ligation had probably not been successful. Therefore PCR was performed, but the primers we used, were not appropriate. Chelate chromatography. We used native conditions (DOC, washing buffers without guanidine ...) for P2 peptide. Denaturing conditions (buffers with guanidine) were used for P4. We also tried using denaturing conditions on both P2 and P4 with elution by 60% acetonitrile/TFA. We clearly saw that this elution mixture washed nickel cations from the column, because it turned white. We measured elution fraction spectra from elution of P4 with denaturing buffers but we did not find any protein there. It is possible that there was a problem with the buffers or that P4 eluted before we even applied elution buffer. We did see an absorption peak at 280 nm when measuring native P2 elution fractions. Inoculum - P4 and "double construct".

August 11th, 2009
PCR for amplification of certain sequences from previously made plasmids to make the following BioBricks: ccdB, gyrB, KSI-DP PCR product and vector with His-tag at the N-terminus of the construct were cut with the same enzymes and then ligated. PCR product p53 was cut out of vector with His-tag at the N-terminus of the construct and ligated into front vector. Transformation of T1W_EEE and T1W_AEA, previously ligated into vector with His-tag at the N-terminus of the construct, into DH5a. Fermentation of P4 and "double construct". We tried to crosslink dimtetra into a protein membrane by filtering it through a filter paper, so dimtetra remains on it, and then crosslinking it. We also tried it by heating the filter paper with dimtetra on it, cooling it and then crosslinking it. After crosslinking it, we dipped the filter paper in ethanol. It seemed as if there was a layer on the paper in the first case (but nothing conclusive) and in the second case we noticed some flakes floating on the surface of ethanol. We will continue with these experiments in the future.

August 12th, 2009
We performed control restriction which showed that this time vector without His-tag was prepared successfully. Vectors T1WDDD_T1WKKK and TA_TB_TC_ELST were isolated and cut with appropriate enzymes to obtain trimerization domains. Colony PCR of Z1 and AUP were put on gel. Because the negative control for Z1 was positive, we cannot be sure, whether Z1 really is in vector with His-tag at the N-terminus of the construct. Ligation of MSC-P-ccdB into vector with His-tag at the N-terminus of the construct. Dot blot of dimtetra solution and double construct, P2 and P4 lysates. 10 ul droplets were vacuumed through a nitrocelulose membrane. The membrane was then blocked in 5% milk. The membrane was shaken in a bag with primary anti-His-tag Abs dissolved in 1% milk for 1h on 37 °C. The membrane was then washed and incubated with secondary Ab (with peroxidase). After washing, a color reagent was added. Dimtetra - the positive control was weak and we did not see anything else. Next step - Western Blot.

August 13th, 2009
PCR of negative control for Z1, that was positive yesterday, was repeated but result was the same again. Transformation of vectors with MCS-P-ccdB into DB3.1 cells. Manual plasmid isolation of T1W_EEE and T1W_AEA and control restrictions. Vectors T1WDDD_T1WKKK and TA_TB_TC_ELST were cut again, because concentrations of isolated fragments were very low. Control restriction of vectors with His-tag at the N-terminus of the construct, containing P1_H1_P2, P1_GCN_P2 and cYFP showed that only vector with P1_H1_P2 was ligated successfully. This vector was isolated with GeneJet PlasmidMiniprep kit. The other two ligations were performed once again. Cultures of Viktor+P6P4P2 and Viktor+GyrB in BL21 were made and shaken until late afternoon. P1, P3 and P4 were lysed in DOC, sonicated and then centrifuged. We ran two SDS PAGE gels. One gel was stained with Coomassie and the other was used for Western blot. After transferring, the membrane was being blocked in 5% milk overnight in a refrigerator. Photos will be taken tomorrow.

August 14th, 2009
Ligation form yesterday (MCS_P_ccdB) --> there were a lot of colonies on backligation plate :( Control restrictions of vectors with T1W_EEE and T1W_AEA were put on gel, but the figure was not clear enough. We will repeat control restriction. We performed control restriction of P1H1P2 construct and KSI-DP in vector with His-tag at N-terminus of the construct, which showed that chosen colonies were no good. Ligation of front vector and front insert for P1H1P2 was repeated. KSI-DP-P3/22 construct was cut and ligated in vector with His-tag at N-terminus of the construct. Fermentation of dimtetra, GyrB and "triple construct" (GyrB and "triple construct" were transformed into our cells and other BL21. Western blot: Membrane was blocked some more on room temperature. Primary Ab (anti-His) were diluted in 1% milk (+TBS) and the membrane was shaken in a bag with the mentioned solution for 1h30min on room temperature. The membrane was washed with TBS and then incubated with secondary antibodies for 1h30min and washed again. We used "femto" reagent for detection. Films were developed.We clearly saw both d53 bands and also some higher ones. Nothing else was seen, except a signal from P4 lysate (lane 7), but the apperent MW seems too high.

August 16th, 2009
We inoculated colonies with vectors, that have His-tag on N-term. and hopefully inserted T1W_EEE and T1W_AEA in order to make new control restrictions.

August 17th, 2009
Manual isolation of plasmids (T1W_EEE and T1W_AEA), that were inoculated yesterday and control restrictions seem to be successful but in order to be sure, GeneJET Plasmid Miniprep Kit isolation and control restriction will be performed. GeneJET Plasmid Miniprep Kit isolation of vectors (with His-tag on N-term.) with inserted Z1 and AUP. Control restrictions seem to be successful. Because there were lots of colonies on backligation plate (on August 14th), we inoculated colonies with MCS-P-ccdB inserted into vector with His-tag on N-term. Dialysis of dimtetra from 6M GvHCl/50 mM TRIS/pH7 to 3L MQ. Lysis (with DOC) and sonication of triple construct (P6P4P2) ---> will be put on SDS PAGE. We ran two PAGEs again - one for Coomassie and the other one for Western. We believe we can see GyrB but only Western blot can confirm it because some constitutive bands are also in close vicinity. We repeated colony PCR for KSI-DP with different set of colonies. Two colonies that appeared OK were later inoculated. We performed PCR to obtain APH1. The reaction was not successful, therefore it was repeated. P1H1P2 and KSI-DP-p2/33 constructs were transformed in DH5a cells.

August 18th, 2009
We isolated (GeneJET Plasmid Miniprep Kit) vectors with His-tag on N-term. with T1W_EEE and T1W_AEA inserted. Control restriction. Manual isolation of vector with His-tag on N-term. and MCS_P_ccdB inserted. Control restriction. Western blot was completed. This time we used ECL reagent first. Then we tried femto also. We can clearly see dimtetra and GyrB. The presence of GyrB shows that Viktor works, but the cells also seem to have problems with producing P6P4P2 (and single P1-P8 and double construct for that matter). We discovered that unfortunately PCR we performed yesterday was unsuccessful again. So, we decided trying to conduct the reaction in 2 stages. Today we set the first stage. Colony PCR was performed on colonies containing P1H1P2 and KSI-DP-P3/22 constructs. One colony containing P1H1P2 construct was inoculated in Mini-prep. None of the colonies contained KSI-DP-P3/22 construct. Constructs: TA, TB, TC, ELST, T1W_DDD and T1W_KKK were cut and then ligated in vector with His-tag at N-terminus of the construct. Vector with His-tag at N-terminus of the construct containing KSI-DP was isolated from Mini-preps. Vector with His-tag at N-terminus of the construct containing KSI-DP was cut for front insert and Vector with His-tag at N-terminus of the construct containing KSI-DP containing P6P4P2 for front vector. The reaction was not successful. BL21 cells were transformed with vector with His-tag at N-terminus of the construct containing KSI-DP.

August 19th, 2009
KSI-DP construct was innoculated for "small" fermentation. Dialysis of dimtetra from 6M GvHCl into MQ. Control restrictions from yesterday were put on gel, which showed us that T1W_EEE, T1W_AEA and MCS-P-ccdB are ligated in vector with His-tag on N-term. successfully. We transformed T1W_EEE and T1W_AEA into BL21 cells. Two clones of MCS-P-ccdB were sent to be sequenced. PCR we performed yesterday to obtain APH1 was put on gel. PCR product was later isolated from gel. Then we set the second stage of the reaction. We performed colony PCR to check the presence of KSI-DP-P2/33 in vector with His-tag at N-terminus of the construct. Vector with His-tag at N-terminus of the construct containing KSI-DP was cut for front insert and Vector with His-tag at N-terminus of the construct containing KSI-DP containing P6P4P2 for front vector again. The reaction was also unsuccessful. DH5a cells were transformed with ligation mixtures from yesterday.

August 20th, 2009
Vector with His-tag at N-terminus of the construct containing P1H1P2 was isolated from Mini-prep. Vector with His-tag at N-terminus of the construct containing KSI-DP and vector with His-tag at N-terminus of the construct containing P3P4H1 were cut to obtain front insert and front vector, this time using different enzymes. Mini-fermentation of KSI-DP VIKTOR Goal of this experiment is to determine, if vector VIKTOR is working with newly inserted KSI domain. KSI -DP inoculum from overnight LB medium was transferred in 100ml LB medium with ampicillin, so the new OD600 was 0.2. After 2.5 hours fermentation (when OD600 was 1.1) KSI product was induced with 1mM IPTG. Over 4 hours of production, cell were harvested by centrifugation (15 minutes and 5000 rpm) KSI-DP pelets were lysed and sonicated. Then lysate was centrifuged (10 minutes at 12000 rpm). Supernatant was segregated from pellets and stored at -80 °C. Protein pellet was dissolved in 6M guanidine hydrochloride. Once again dissolution was centrifugated. Pellet was stored at -80 °C, supernatant with possible KSI was put in dialysis (dialysis tube 0.5 ml against 1000 ml of MQ) overnight. Vector with His-tag on N-term. containing MCS-P-ccdB was isolated using GeneJET Plasmid Miniprep Kit in order to be sequenced (not sent yet). Vector with His-tag on N-term. was digested by BspEI/PstI, put on gel and isolated (MinElute Gel Extraction Kit) in order to be prepared for insertion of four glutamates between SpeI and terminator, that is in front of terminator. Vector with His-tag on N-terminus of the construct containing P1H1P2 was isolated from mini-prep. Front vectors P2P6H1 and P3P4H1 were isolated from gel. Vector with His-tag on N-terminus of the construct containing P3P4H1 and vector with His-tag on N-terminus of the construct containing KSI-DP were cut for front vector and front insert respectively and later isolated from gel. Front vector P3P4H1 and front insert KSI-DP were then ligated. Electrophoresis showed that PCR we performed to obtain APH1 was not successful. BL21 cells wee transformed with vector with His-tag on N-terminus of the construct containing P1H1P2 and vector with His-tag on N-terminus of the construct containing P1GCNP2.

August 21st, 2009
We inserted four glutamates in front of terminator in vector with His-tag at N-term. of the construct by ligation and transformed it into DB3.1 cells. Fermentation of T1W_AEA and T1W_EEE protein T1W_AEA 0.5l fermentation, inoculum OD600: 0.2, induced at OD600: 0.92 with 1mM IPTG, 4 hours fermentation, final OD600 was 3.1 T1W_EEE 0.5l fermentation, inoculum OD600: 0.2, induced at OD600: 0.99 with 1mM IPTG, 4 hours fermentation, final OD600 was 2.9 Cells were centrifuged (5000 rpm, 15 min) and stored at -80°C. Dialysed KSI-DP precipitated. Precipitate was separated from the soluble fraction. We did an SDS PAGE with KSI-DP and some other samples but most likely the samples were not prepared well. -GyraseB band (26kDa) is well seen. This confirms presence of GyrB protein in pellet after centrifugation of lysed cells. Majority of GyrB protein is in supernatant of lysed cells as we have proved on previous SDS (figure 12) and western blot (figure 13). Vector VIKTOR is working properly. -There is no Triple construct P6P4P2 (13.3kDa) band on gel. We have no production of this protein. Optimization of fermentation and production process is needed. -In all four fractions of lysate KSI-DP protein is missing. We assume that something is wrong with DNA sequence of KSI_DP. -P2/P4 There are no P2 and P4 protein present in lysed cells. Optimization of fermentation and production process is needed. Vectors with His-tag on N-terminus of the construct containing TA, TB, TC, ELST, KSI-DP-P3/22, P3P5 were isolated. Vectors containing TA, TB and TC were cut for front vector and front insert. Vectors containing ELST, P3 and P3P5 were cut for front vector. Later they were cut from gel. We performed control restriction on vectors with His-tag on N-terminus of the construct containing cYFP, T1W_DDD and T1W_KKK. Vectors containing correct inserts were then isolated.

August 22nd, 2009
We performed colony PCR to confirm the presence of KSI-DP-P3P4H1 in Vector with His-tag at N-terminus of the construct. We discovered that ligation was not successful. Vectors with His-tag at N-terminus of the construct containing P1 and P3 were isolated. Vector containing P1 was then cut for front insert. Front vectors TA, TB, TC, P3, P1, ELST, P3P5 and front inserts TA, TB, TC were isolated from gel.

August 24th, 2009
We inoculated 10 colonies of DB3.1 cells, containing four glutamates in front of terminator in vector with His-tag at N-term. in order to perform isolation and control restriction tomorrow. Vectors with His-tag at N-terminus of the construct with changed restriction sites at the ccdB domain (MCS-P-ccdB) were sent to be sequenced. We repeated restriction of vector with His-tag at N-terminus of the construct containing KSI-DP, to obtain KSI-DP front insert and then ligation of KSI-DP (front insert) and Vector with His-tag at N-terminus of the construct containing P3P4H1 (front vector). Front vectors TA, TB, TC, P3, P1, ELST, P3P5 and front inserts TA, TB, TC, P1 were ligated and then transformed into DH5a cells. Fermentation of P1-H1-P2 and P1-GCN-P2 trimer proteins P1-H1-P2 0.25l fermentation, inoculum OD600: 0.1, induced at OD600: 0.5-0.6 with 0.4mM IPTG. Fermentation 2 hours, OD600: 2.5, 25ml of cells were stored. Fermentation 4 hours, OD600: 3.5, 220ml cells were centrifuged and stored at -80°C P1-GCN-P2 0.25l fermentation, inoculum OD600: 0.1, induced at OD600: 0.5-0.6 with 0.4mM IPTG. Fermentation 2 hours, OD600: 2.7, 25ml of cells were stored. Fermentation 4 hours, OD600: 4.1, 220ml cells were centrifuged and stored at -80°C Samples (25 ml) were also taken before induction of P1-H1-P2 and P1-GCN-P2. Stored at -80°C. SDS-PAGE of KSI-DP lysate gave final evidence of absence KSI-DP protein in cells. We also got report of false sequence of KSI domain. On SDS PAGE we can see absence of T1W_EEE (9.5kDa) band in supernatant and pellet. Contrary T1W_AEA (8.5kDa) has strong band, which confirms production of this protein.

August 25th, 2009
Vector with His-tag at N-term. containing four glutamates in front of terminator was isolated, control restriction was performed successfully. Colony PCR was performed on colonies transformed with vectors with His-tag at N-terminus of the construct containing P1P3P5, TaP1, TBP3, TCP5, P2TA, P4TB, P6TC.

August 26th, 2009
Vector with His-tag at N-term. containing four glutamates in front of terminator was isolated with GeneJET Plasmid Miniprep Kit and sent to be sequenced. We performed PCR (with temperature gradient); the template: only primers for APH1.. We inoculated 10 mL LB+antibiotic for miniprep with vector with His-tag on N-term. containing different inserts: p53, foldon, gyrB, CutA1 and vector alone. Control restrictions showed that nor KSI-DP neither KSI-DP-P2/33 are present in vector with His-tag at N-term. We figured out why and are about to clone inserts into vector properly.Control restrictions showed that nor KSI-DP neither KSI-DP-P2/33 are present in vector with His-tag at N-term. We figured out why and are about to clone inserts into vector properly. Control restriction was performed on vectors with His-tag at N-terminus of the construct containing P1P3P5. One clone was later isolated from mini-prep. Constructs P1H1P2, P1P3P5, cYFP and KSI-DP-P2/33 were sent for sequencing.

August 27th, 2009
Vectors with His-tag at N-terminus of the construct containing P1P3P5 and P6P4P2 were cut for front vector. Front vector P1P3P5 was later isolated from gel. PCR products of APH1 were put on gel and isolated. We isolated the following plasmids by GeneJET Plasmid Miniprep Kit: vector with His-tag on N-terminus containing different inserts: p53, foldon, gyrB, CutA1 and vector alone and performed restrictions, using appropriate enzymes, put them on gel. We cut out restricted vectors and fragments respectively to be purified tomorrow. We repeated PCR to obtain nYFP, KSI-DP, KSI-DP-P3/22. Products were isolated from gel. PCR for nYFP was later repeated at different annealing temperatures. Constructs TAP1, TBP3, TCP5, P2TA, P4TB, P6TC and p53 were sent for sequencing. In order to produce vector with His-tag at N-term. containing KDI-DP or KSI-DP-P2/33 we cut PCR products 2 and 12 (KSI-DP and KSI-DP-P2/33) and vector with His-tag at N-term. with enzymes XbaI and PstI and then ligated each PCR product into the vector. Ligation products of this stage are vectors with His-tag at N-term. containing KDI-DP or KSI-DP-P2/33 but lacking terminator sequence. Ligation products was transformed into DH5a cells.

August 28th, 2009
After the transformation that was performed yesterday, only the colonies of bacteria transformed with vectors with His-tag at N-term. containing KDI-DP grew on LB plates. Some of them were inoculated for miniprep. Ligation of KSI-DP-P2 into the vector was set again and ligation product was transformed into DH5a cells. Restricted vectors and fragments (Viktor with p53, foldon, GyrB, CutA1 ...) were purified from gel. APH1 was restricted NgoMIV/SpeI and ligated into vector with His-tag on N.term. Ligation was set for various constructs - GyrB+p53, GyrB+foldon, GyrB+CutA1, Gyr-SG (longer linker), foldon+p53. APH1 was set to be ligated into Viktor. nYFP PCR products were put on gel and later isolated from gel. PCR ligation to obtain nYFP was repeated. Several flasks with 10 mL LB were inoculated with colonies containing vectors with His-tag at N-terminus of the construct with P6P4P2.

August 29th, 2009
Control restriction was performed to confirm the presence of P6P4P2 constructs in vector with His-tag at N-terminus of the construct. Vector was later isolated. APH, ligated into vector with His-tag on N.term, was transformed into DH5a cells. Vector with His-tag at N-terminus of the construct containing KSI-DP but lacking the terminator sequence was isolated. Control restriction was performed to confirm the presence of KSI-DP construct in vector with His-tag at N-terminus (lacking terminator sequence) of the construct.

August 30th, 2009
Some clones containing several constructs, were inoculated for miniprep.

August 31st, 2009
Construct foldon+p53, inserted into vector with His-tag on N-term. (=construct no. 29) and vector with His-tag on N-term. and APH1 were isolated and restricted: control restriction of construct no. 29 and restriction of APH1 in order to be used to make construct no. 28. Vector with His-tag at N-terminus of the construct containing KSI-DP-P2/33 but lacking the terminator sequence was isolated. Control restriction was performed to confirm the presence of KSI-DP-P2/33 construct in vector with His-tag at N-terminus (lacking terminator sequence) of the construct. Vectors with His-tag at N-terminus of the construct containing KSI-DP, and KSI-DP-P2/33 but lacking the terminator sequence and Vector with His-tag at N-terminus of the construct were cut with EcoRI and SpeI. KSI-DP (FI) and KSI-DP-P2/33 (FI) were ligated into the cut vector resulting in vectors with His-tag at N-terminus of the construct containing KSI-DP or KSI-DP-P2/33. Vector with His-tag at N-terminus of the construct containing P6P4P2 was cut for front vector and later isolated from gel. Products of PCR ligation (August 28th) were also put on gel and then isolated. Front vectors P1P3P5 and P6P4P2 were ligated with front insert KSI-DP.

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