Template:Vilnius-Lithuania/JS/Experiments
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{
name: "June 22 - June 28",
from: 22,
to: 28,
d: "Helimerase synthesis: pETDuet-10xHis-MBP-TEV-StrepII plasmid (hereinafter pETDuet plasmid) was transformed into <i>E. coli</i> ER2566 competent cells. After purifying two cloning strategies was performed. TteUvrD as well as BstPol were cloned into the plasmid employing two methods that differed in the order of gene insertion, i.e. using one method TteUvrD was the first one to be inserted into pETDuet first MCS (dubbed as pETDuet1 or PD1) and using another method BstPol was the first one to be inserted (pETDuet2 or PD2) into the second MCS.
Next day interim ligates were once again digested and the remaining gene was inserted. Then, the complete ligates were transformed into an E. coli ER2566 strain. Alas, ligation was not successful because there were no colonies after night incubation. Ligation was repeated again and plasmids were again transformed into the same strain, however as per last time no colony growth was observed. It was decided to attempt a different cloning technique before amplifying more of the TteUvrD and BstPol via PCR.
LuxS synthesis: luxS gene was cloned into pET28a(+) vector, resulting ligate was transformed into E. coli DH10B strain. After transformation colony PCR was performed to select suitable colonies. Select colonies were grown overnight, their plasmids purified. Restriction analysis of purified plasmids was performed. Only one sample displayed good results. According to these results, this plasmid was chosen for further experiments.
Pfs protein synthesis: pfs gene was ligated into pET28a(+) plasmid ant resulting construct transformed into E. coli DH10B strain. cPCR was performed and suitable colonies was transferred to liquid medium for plasmid cloning. Next, pET28a(+)-pfs plasmid was purified and restriction analysis carried out - all results were positive. Then the plasmid was transformed into E. coli DH10B and E. coli BL21(DE3) strains.
Ep promoters: PCR amplification of lsrACDBFG, EP01, and EP14 promoters. Ligation of aforementioned promoters and sfGFP protein into a pSB1C3 plasmid. Agarose gel electrophoresis revealed that restriction was not successful.
", p: "GldJ synthesis: Flavobacterium columnare, F. branchiophilum and F. psychrophilum were cultured in liquid media. PCR was carried out in order to amplify GldJ (gliding motility lipoprotein) gene, however the experiment was unsuccessful.
VHSV glycoprotein synthesis: pfx-7 vector and VHSV (viral hemorraegic septicemia virus) insert were digested with restriction enzymes and ligated together. Ligates were transformed into E. coli DH10B strain, however due to human error the experiment was a failure. Procedure was repeated and transformants were subjected to colony PCR. cPCR brought no usable results and was repeated. The repeated cPCR showed only the presence of <200bp fragments, this was interpreted as a ligation failure.
" },
{
name: "June 29 - July 5",
from: 29,
to: 5,
cross: 30,
d: "Helimerase synthesis: After another transformation using fresh ligates was unsuccessful, pETDuet plasmid agarose gel electrophoresis was performed to investigate whether or not it was the correct intact plasmid. No plasmid was observed to be present, possibly due to its low concentration. A decision was made to employ different cloning strategies as well as to repurify the pETDuet plasmid again. pETDuet was purified from E. coli DH10B transformants. TteUvrD and BstPol gene fragments were ligated into the pETDuet plasmid using the same cloning strategy utilized last week.
Ligates then were transformed into E. coli DH10B strain and incubated overnight. After positive transformation results, colony PCR was performed to determine which colonies contain our specific constructs. Selected colonies were incubated and their plasmids were purified. After purification, the obtained pETDuet-TteUvrD (PD-T) and pETDuet-BstPol (PD-B) plasmids were digested and new inserts (TteUvrD and BstPol respectively) were cloned into them. PD-TB and PD-BT ligates were then transformed into an E. coli DH10B strain and left overnight.
Helicase dependent amplification: Genomic DNA of Flavobacterium columnare and F. Psychrophilum was extracted. Fragments of 16S rRNA gene chosen for detection were amplified via PCR. Gold nanoparticles were prepared using Turkevich method with modifications.
", t: "LuxS synthesis: Small-scale LuxS induction was attempted. Results were satisfactory - protein was found to be well expressed and soluble after mere 3 hours of induction. Additional pET28a(+)-luxS plasmid was purified.
Pfs protein synthesis: Purified plasmid from the DH10B strain was prepared for sequencing, meanwhile BL21(DE3) strain was moved onto induction experiment.
Anderson promoters: Lyophilized five J61002 plasmids which contain different strenght Anderson promoters (J23100, J23103, J23105, J23111 and J23114) from 2019 DNA Distribution Kit were solubilized. These five promoters were transformed into E. coli DH10B strain. Unfortunately, this bore no results. Next, we attempted the same thing with the same promoters from the 2016 DNA Distribution Kit, however once again the transformation was unsuccessful. Quite some troubleshooting followed.
", p: "GldJ synthesis: Alternative cultivation method using TYES agar was applied, however the PCR was still unsuccessful. After that, PCR was modified to contain SuperFi™ GC Enhancer and PEG 4000. Finally, some positive results were observed.
VHSV glycoprotein synthesis: pfX-7 VHSV construction procedure was repeated, however no ligate band was seen in agarose gel electrophoresis. In addition, after running pure VHSV fragment gel, again no bands were observed. Later, it was discovered that liophylised VHSV fragment was not properly dissolved. After preparing a fresh solution, a band containing our fragment appeared. Cloning process was repeated, however post-transformation colony PCR revealed the insert did not embed correctly.
" },
{
name: "July 6 - July 12",
from: 6,
to: 12,
d: "Helimerase synthesis: Restriction analysis of PD-TB and PB-BT plasmids was performed. Transformation of eligible PD-helimerase plasmids into an E. coli BL21(DE3) strain followed. The next day, Helimerase induction was performed, however SDS-PAGE electrophoresis revealed that there was no targeted-size protein. It was decided to sequence the construct. In parallel, entire cloning process was repeated. Regarding to smaller proteins seen in the gel, it was hypothesised that the MBP protein is being cleaved from the TteUvrD gene during this protein synthesis.
", t: "LuxS synthesis: Large-scale (200 mL medium volume) induction was repeated. Post-induction volume was sonified and underwent Ni-NTA chromatography. Desirable fractions were collected and dialysed. Resulting protein concentration was measured using NanoDrop, sample was diluted with 100% glycerol and stored in -20°C.
mazF toxin and mazE anti-toxin system: mazE and mazF gene fragments were amplified via PCR, fragment size was confirmed via DNA agarose gel electrophoresis. mazF fragment was ligated into pSB1A2-sfGFP plasmid. Resulting ligate was then transformed into E. coli DH10B cell line, however, unsuccessfully.
recNp synthesis: recNp PCR amplification. recNp was ligated into pSB1C3-sfGFP vector and unsuccessfully transformed into E. coli DH5α strain. Use of TG1 cell strain delivered positive results. After cPCR selected colonies were growth overnight and their plasmids were purified. Restriction analysis revealed that the cloning process was not successful.
Anderson promoters: After several (DH10B, DH5α, TG1) E. coli strains were tested at low ampicillin concentrations, finally, transformation was found to be successful. This was also confirmed by cPCR results. Plasmids from different strains were purified. Additional Anderson promoters were also transformed under suitable conditions and the plasmids were then purified.
Exolysin gp531 synthesis: pET16-gp531 plasmid was transformed into E. coli BL21(DE3) cell line. After protein synthesis induction, it was discovered that the exolysin was poorly soluble, thus it was decided to attempt the induction in using different E. coli strain.
", p: "GldJ synthesis: Gradient PCR was carried out using ColF and ColRv primers in order to determine optimal PCR conditions. Once PCR conditions were optimized, PCR amplification of GMP gene was reattempted. However, desirable fragments were not obtained. Despite this, alternative fragments of ~1000bp and ~2900bp were amplified, since it was believed that the ~1000bp fragment was DnaJ protein gene, which could be used as an alternative to GMP. Fragments were ligated into pET-28a(+) vectors and trasnformed into E. coli DH10B strain. Transformation was unsuccessful.
VHSV glycoprotein synthesis: Restriction analysis of pfX-7 was performed however gel electrophoresis provided no visible bands. It was postulated that at some point plasmid solution was contaminated with degrading substances. In addition, further necessary plasmid manipulation steps were discovered that may have caused previous lack of success. After the method adaptation transformants were overgrown. Experiment was repeated. The next few days were chock-full of dissatisfying colony PCR results.
" },
{
name: "July 13 - July 19",
from: 13,
to: 19,
d: "Helimerase synthesis: Continuation of repeat cloning and helimerase expression experiments: PD plasmid digestion, ligation with TteUvrD/BstPol fragments, transformation, etc. Unfortunately, induction was once again unsuccessful. It was decided to co-express pETDuet plasmids containing different genes in it (PD-TteUvrD and PD-BstPol). Thus, PD-T and PD-B plasmids were transformed into E. coli BL21 (DE3) strain and incubated overnight.
Helicase dependent amplification: Extraction of genomic F. branchiophilum and F. psychrophilum DNA.
", t: "mazF toxin and mazE anti-toxin system: J61002-J23104 plasmid was purified, and ligated with mazE.
recNp synthesis: After failed cloning, construct assembly was repeated with a stock recNp fragment. recNp ligated with a pSB1A2-sfGFP plasmid and transformed into DH10B strain. Restriction analysis verified correct cloning outcome. After cultivation of select colonies, their plasmids were purified. In addition, recNp-sfGFP plasmid was transformed into a DH10B cell line.
Anderson promoters: Purified plasmid concentration was intolerably low, thus, plasmids were retransformed into a DH10B strain for additional cloning. sfGFP was cloned into the plasmids containing different Anderson promoters.
Exolysin gp531 synthesis: Constructs were transformed into E. coli Rosetta (DE3) and ArcticExpress (DE3) strains and induction procedure was repeated.
", p: "GldJ synthesis: Bacterial glycerol stocks of F. columnare, F. branchiophilum and F. psychrophilum were prepared and stored at -80°C. Restriction analysis of ~1000 bp and ~2900 bp fragments was performed in order to determine whether or not DnaJ protein gene was present in any of them. Repeated gradient PCR, reoptimised PCR procedure. After the most suitable conditions for PCR were determined, the PCR was repeated. Once again, 1 and 2.9 kbp fragments were obtained, which were inserted into pET28a(+) vector and transformed into E. coli DH10B strain. Transformation was successful only using the 2.9 kbp fragment-containing ligate. Subsequent colony PCR confirmed the results.
VHSV glycoprotein synthesis: After some research further adaptations to the experiment plan were added, in order to troubleshoot persisting issues. Namely, for the time being, pfX-7 plasmid was replaced by pJET1.2 whilst working with DH10B strain. Also, for pfX-7 cloning GM119 strain was employed. Sadly, pJET-VHSV transformation bore no results. After repeat transformation it was successful. pJET-VHSV was purified from select colonies and transformed into GM-119 cell line and this again went as planned. Then, after purifying the plasmid from GM-119, VHSV fragment was cut out and ligated back into pfX-7. Resulting ligate transformed back into GM119. cPCR revealed that the latter transformation was unsuccessful.
" },
{
name: "July 20 - July 26",
from: 20,
to: 26,
d: "Helimerase synthesis: Transformant colony PCR revealed no TteUvrD gene containing plasmids were present in the bacteria. Due to the outcome, alternative helimerase synthesis strategies were employed: first, we attempted to express the proteins separately and fuse them after they were expressed, and second, we attempted to clone BstPol into a different plasmid and then co-express the two genes. First method proved to be fruitless: SDS-PAGE electrophoresis revealed no BstPol protein present in the solution. Thus, it was opted to clone BstPol into a pACYC plasmid whose ORI region matches with the one of pETDuet's allowing for simultaenous co-expression of the two genes from different plasmids in the same cell.
After pACYC-HS and pACYC-SH plasmids were amplified and purified from E. coli DH10B cells, BstPol was ligated into both variants and resulting constructs were transformed into an E. coli DH10B strain. Transformation was unsuccessful and thus repeated. TteUvrD synthesis from the second cloning attempt was also unsuccessful - restriction analysis showed that there was something wrong with the gene fragment. It was elected to clone TteUvrD into a pET28a(+) plasmid while also reattempting the cloning into PD plasmid. cPCR of pACYC-containing colonies was performed, suitable colonies were selected for further experiments, their plasmids purified and restrictively analysed. pACYC-HS-BstPol plasmid transformed into E. coli BL21 (DE3) strain.
Helicase dependent amplification: PCR electrophoresis revealed that all the primers used were suitable for amplification of fragments containing the probe hybridisation sites. Next HDA was attempted, results showed that in order to amplify F. columnare fragment further optimization of HDA parameters was needed.
", t: "Exolysin gp531 synthesis: Protein synthesis and extraction successful.
LuxS synthesis: luxS gene was cloned into a pET28a(+) vector, resulting ligate was transformed into an E. coli DH10B strain. After transformation colony PCR was performed to select suitable colonies. Select colonies were grown overnight, their plasmids purified. Restriction analysis of purified plasmids was performed. Only one sample displayed good results. Regarding these results, this plasmid was chosen for further experiments.
lysECD7 construct: lysECD7 fragment amplified via PCR and ligated into A104-sfGFP vector. Resulting ligation mixture transformed into an E. coli DH10B cell line. Transformation was not successful. It was opted to repeat the process with increased antibiotic concentration. A good amount of colonies were observed on the agar plate, however, cPCR revealed the transformation to be a failure. Later it was discovered that there was an error in the lysECD7 sequence we had received.
", p: "GldJ synthesis: pET28a(+)-fragment(~2900bp) plasmid purification was performed. Restriction analysis using HindIII and Eco32I restriction enzymes followed. Additionally, PCR with F. branchiophilum and F. psychrophilum genomic DNA was carried out with a variety of enhancers. pET28a(+)-fragment(~2900bp) plasmid was once again transformed into an E. coli DH10B strain for further amplification.
" },
{
name: "July 27 - August 2",
from: 27,
to: 2,
cross: 31,
d: "Helimerase synthesis: Induction of BstPol was attempted, albeit unsuccessfully. Restriction analysis of TteUvrD and BstPol containing constructs was performed. No conclusions could be drawn from the results. Because of this it was decided to wait for sequencing results.
Helicase dependent amplification: Due to ineffective efforts to optimise HDA of F. columnare fragment PCR with frozen bacteria was performed. This proved to be of miniscule utility. After many attempts to optimise the HDA protocol, HDA of F. columnare was finally somewhat successful, however further optimisation was still required. HDA using F. psychrophilum gDNA was succesful without major modifications of the protocol.
", t: "mazF toxin and mazE anti-toxin system: J61002-J23110 (hereinafter A110 et cetera), A115, A118 plasmids ligated with mazE. A101, A104 plasmids ligated with mazF. Ligation products transformed into DH10B strain. A115 and A118 plasmid transformation was successful. The restriction analysis showed positive results. In addition, through blood, sweat and tears A101-/104-/110-/115-sfGFP plasmids were constructed, which were then subsequently ligated with <i> mazF </i>and successfully transformed into DH10B strain. A101-/104-/110-sfGFP-mazF constructs were ligated with either EP01 or lsrACDBFG and transformed into DH10B strain. Overall, further progress towards J23AXXX-sfGFP-EP01/EP14/lsrACDBFG-mazF construct progress was made.
", p: "VHSV glycoprotein synthesis: Although the transformations were successful (as indicated by cPCR results) plasmid extraction from gel was not. Repeat extraction yielded more positive results, following restriction analysis also indicated things were in accord with our expectations. Unfortunately, yeast cells died shortly after, thus the transformation had to be repeated.
GldJ synthesis: After sequencing it was discovered that fragment from gDNA PCR was not the one we needed. Since we were not able to synthesize GldJ fragment from bacteria gDNA, we decide to purchase full GldJ sequence.</b>"
},
{
name: "August 3 - August 9",
from: 3,
to: 9,
d: "<p>Helimerase synthesis: While waiting for the sequencing results, western blot, using Anti-MBP primary antibodies, was performed in order to see whether or not MBP-tag as well as TteUvrD protein degrade. Results showed that both the tag and the protein degrade during protein synthesis process. Awaited sequencing results revealed that all sequenced plasmids contained mutations. However, as a last resort some plasmids were still transformed into a variation of E.coli strains and protein induction was attempted in differing conditions. According to SDS-PAGE electrophoresis analysis BstPol induction was a failure. TteUvrD-containing fractions were observed in a Rosetta (DE3) strain. After sonication, the highest amount of TteUvrD protein was observed in the precipitate fraction. It was decided to reattempt TteUvrD cloning process, given no mutations arise this time around.Helicase dependent amplification: Ongoing HDA protocol optimisation.
", t: "mazF toxin and mazE anti-toxin system: A117/A118-mazE-mazF constructs prepared. J23110-/104-/101-sfGFP-mazF-lsrACDBFG constructs assembled. J23110-<i>mazF-mazE</i>-ep01/-ep14 constructs assembled. J23117-/118-mazF-mazE-ep01/-ep14/-lsrACDBFG constructs assembled.
", p: "VHSV glycoprotein synthesis: Transformation was successful, selected colonies were transferred to liquid medium. Next day glycoprotein synthesis induction followed. Synthesis was successful, SDS-PAGE electrophoresis revealed that the protein is soluble, albeit its concentration is quite low.
" },
{
name: "August 10 - August 16",
from: 10,
to: 16,
d: "Helimerase synthesis: Repeat cloning of BstPol into pACYC-HS and of TteUvrD into pETDuet. Resulting ligates transformed into E. coli DH10B. Transformation was successful. cPCR performed and desirable colonies were chosen for sequencing.
Helicase dependent amplification: Ongoing HDA protocol optimisation.
", t: "mazF toxin and mazE anti-toxin system: A110-mazE-mazF-lsrACDBFG ligation repeated. A115-mazE-mazF ligation repeated. Both were successful. A104-pSB1C3-mazE construct assembled ant transformed successfully into a DH10B strain. Further on, A104-pSB1C3-mazE-mazF construct was produced and cloned into DH10B cells.
", p: "VHSV glycoprotein synthesis: a 100 mL of S. cerevisiae AH22-214 culture for an induction experiment was prepared. The synthesized VHSV protein was extracted using glass beads. Protein purified using 6xHis tag chromatography. SDS-PAGE electrophoresis revealed, however, that the chromatography was unsuccessful. An alternative 214Δpep strain was chosen for further experiments due to suspicions that peptidases produced by 214 strain could be degrading the protein. Unfortunately, the protein purification process required some further optimisation.
" },
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name: "August 17 - August 23",
from: 17,
to: 23,
d: "",
t: "",
p: ""
},
{
name: "August 24 - August 30",
from: 24,
to: 30,
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name: "August 31 - September 6",
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to: 6,
cross: 31,
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p: ""
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name: "September 7 - September 13",
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name: "September 21 - September 27",
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name: "September 28 - October 4",
from: 28,
to: 4,
cross: 30,
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name: "October 5 - October 11",
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name: "October 19 - October 25",
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d: "",
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p: ""
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{
name: "October 26 - November 1",
from: 26,
to: 1,
cross: 31,
d: "",
t: "",
p: ""
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