Team:DeNovocastrians/Notebook
Aakash: Media preparations. Overnight cultures of Rhodococcus strain 9, Rhodococcus strain 33, E. coli and ADP1. Growth curves in LB media/M9+(15g/L)Benzoate media. E.coli::PTTQ18/PETDUET/PACYC plasmid extractions.
Ruby: Growth curves of Rhod 9, Rhod 33 and ADP1 in LB and M9 media. Primer design. Prepared PACYC Duet strain DH5α, PetDuet strain DH5α and PTTQ18 strain DH5α for future plasmid preparations. Streaked PACYC and PetDuet onto 30ug/ml CAM agar plates. Streaked PTTQ18 onto 100ug/ml AMP agar plate. Prepared overnight cultures in M9 media - pipette inoculated 100ug/ml Amp M9 solution with PTTQ18, pipette inoculated 50ug/ml CAM M9 solution with PACYC and PetDuet.
Darcy&Cate: Strain 33&9 were cultured for 2days to allow for genomic DNA extraction.Extraction of Rhod 9 and Rhod 33 genomic DNA by using the DNeasy Ultraclean microbial kit. PCR was undertaken on the extracted gDNA, strain9&33 gDNA was diluted down to 10ng/uL Rhod Strain 9
g
Aakash: B.subtilis(168), E.coli(BW25113), Synechocystis overnight cultures. B.subtilis(168) and E.coli(BW25113) growth curves in 10mM/100mM Benzoate.
Ruby: PCR of Rhod 9 benE, his tagged Rhod 9 benE, ADP1 benE and his tagged ADP1 benE.
Darcy&Cate: A PCR was undertaken on the BenABCD cluster of Strain 9 and E.ferg. This was was repeated twice. And gel electrophoresis was undertaken on all PCR samples
Aakash: Synechocystis PCC 6803 Growth Curve in 10mM/100mM Benzoate.
Ruby: PCR Rhod 9 benE, his tagged Rhod 9 benE, E. Ferg benE. Checked E.Ferg g-block. Ethanol precipitation of Rhod 9 benE and his tagged Rhod 9 benE PCR products.
Darcy&Cate: This PCR was a repeat of the previous week (12/08/2020) with 1 more cycle of PCR. Another PCR was run with lower annealing temp and high GC buffer. Gel were run for all PCR samples.
Aakash: E.coli BL21, E.coli BW25113, B.subtilis, Acinetobacter ADP1, Rhod strain 9, Rhod strain 33 overnight cultures. Growth curves of E. coli BL21, E. coli BW25113, B. subtilis, Acinetobacter baylyi ADP1 in 10mM/100mM/1M Benzoate (Microtiter plate).
Ruby: PCR Cleanup of ADP1 benE and ADP1 benE his PCR products.
Darcy&Cate: PCR clean up was done for the PCR products of ADP1,E.ferg and Rhod St.9. This was follwed by a digestion and ligation reation to clone the PCR products into the PTTQ18 plasmid.
Aakash: Rhod strain 9/Rhod strain 33/E.coli/ADP1/168 overnight cultures. Catechol media prep. Synechocystis subculture. Growth curves of E. coli BL21, E. coli BW25113, B. subtilis, Acinetobacter baylyi ADP1 in 10mM/100mM/1M Benzoate (Microtiter plate). E. coli (BL21)::PTQ118 plasmids extraction.
Ruby: Digests - ADP1 benE and ADP1 his benE into pTTQ plasmid, Rhod 9, Rhod 9 his and E. Ferg benE into pET30a plasmid. Ligation. Transformations of plasmids into competent E. coli cells. Transformed E. coli cells grown on selection plates.
Darcy&Cate The ligation reaction was repeated with antartic phosphatase. A transfromation was attempted but failed. After this, a digestion was done on the restriction enzymes to check function.
Aakash: E. coli BL21, E. coli BW25113, B. subtilis, Acinetobacter baylyi ADP1 overnight cultures and growth curves in serial diluted 1M Catechol (microtitre plate).
Ruby: PCR confirmation of transformations. Overnight cultures of transformed cells. Extracted plasmids from overnight cultures. Ran digests to check for benE in plasmids.
Aakash: Rhodococcus 9, Rhodococcus 33 and ADP1 overnight cultures and growth curves in serial diluted 1.5M Catechol (microtitre plate).
Ruby: PCR of ADP1 benE upstream, benE downstream, benK downstream and benK upstream fragments for knockout PCR. PCR cleanup of products. Overnight cultures of plasmid lab stock cultures: KH142, IG1110 and IG245. Plasmid preps on these overnight cultures.
Darcy&Cate: The cloned plasmid was sent off for sequencing and bioinfomatics of the benABCD cluster was undertaken.
Luke: using ADP1 gDNA(isolated from Ruby “Igem member”), made 3 PCR to isolate fragments from gDNA 0.6ul of gDNA in each reaction, with extension time 70°C for 25 seconds. To isolate CatM binding region, CatM promoter and BenM binding region.
Run on ethdium Bromide gel and CatM promoter and BenM binding region, showed bands at correct length. CatM binding region showed no DNA present in lane.
BenM, GFP, mCherry and CatM gene sequences arrived from Twist.
Created working solutions of all fragments to contain 1ng/ul for all regions, which was used for PCR, to join all fragments together in one reaction.
Run on ethidium bromide gel and contained no bands at desired length
Performed overlap extension PCR, by using 1ul of 1ng/ul concentration for each fragment in PCR. 6 fragments with corresponding primers, to contain overlap regions from primers.
| Reaction | Template | Primer 1 | Primer 2 | Concentration ng/uL |
|---|---|---|---|---|
| 1 | mcherry gene | 1FWD | 1REV | 197 |
| 2 | catm binding region | 2 REV | geneless REV | 23 | 3 | catm binding region | 2 REV | geneless REV | 23 |
| 4 | catm gene | 4FWD | 4REV | 191 |
| 5 | benm gene | 5FWD | 5REV | 42.2 |
| 6 | benm promoter/binding region | 6REV | GENELESS FWD | 60 |
| 7 | GFP gene | 7FWD | 7REV | 44.6 |
Run on ethidium bromide gel
Run on ethidium bromide gel,
each band was at the correct location, although there was other bands and smear present within gel
Aakash: Rhodococcus 9, Rhodococcus 33 and ADP1 overnight cultures and growth curves in serial diluted 1M Benzoate (microtitre plate).
Ruby: PCR on IG245 plasmid amplifying FRT sites for gene knockouts. PCR cleanup of products obtained.
Darcy&Cate: ADP1 and Rhod9 DNA was amplifed with new primers, this was then used in a digestion and ligation reaction.
Luke;Performed overlap extension PCR, by using 1ul of 1ng/ul concentration for each fragment in PCR.
Trying to join mCherry to Catm binding(geneless), mCherry to Catm binding(whole construct), Gfp to BenM promoter(geneless and whole construct), CatM promoter to CatM gene, all fragments for whole construct, all fragments for geneless construct.
Run on 45ul on thyazoile orange gel and all lanes contained nothing(not enough DNA used).
Used remaining 5ul for amplification with corresponding primers for each potentially joined fragments.
Performed gel extractation for GFP-BenM promoter(geneless), GFP-BenM promoter and CatM promote-CatM gene. All other overlap extension PCR did not contain band in the correct position.
Solutions below followed optimization protocol for overlap extension PCR(Hilgarth and Lanigan, 2020).
Trying to join mCherry to Catm binding(geneless), mCherry to Catm binding(whole construct), Gfp to BenM promoter(geneless and whole construct), CatM promoter to CatM gene, all fragments for whole construct, all fragments for geneless construct.
run on tyhazoile orange gel
mCherry-CatM binding (geneless), failed(lane 2)
m-Cherry-CatM binding(whole construct), success, although large amount of smearing and other bands(lane 3)
whole construct, not in correct position, not at correct position, although large amount of smearing and other bands(lane 4)
geneless construct, not in correct position, not at correct position, although large amount of smearing and other bands(lane 5)
from whole construct and geneless construct, removed 5ul from reraction and amplified using primers for whole fragment(hopefully to purify, less smearing, less bands). then run reaction on thiazoile orange.
Same result, large amount of smearing and other bands present.
Gel extracted geneless construct band(lane 2), whole fragment at fully constructed length 4500(although there was no band) and at bright band 3500(lane 3).
Amplified via PCR using just 0.5ul from each solution, then run on gel, no DNA present within lane.
Using potential extracted geneless solution, performed restriction, ligation into pUC19 plasmid and transformed into DH5alpha cells.
Was unsuccessful, no transformations.
Performed overlap extension PCR following optimized protocol(Hilgarth and Lanigan, 2020). Of CatM promoter with CatM binding and BenM promoter with GFP.
Aakash: Rhodococcus 9, Rhodococcus 33 and ADP1 overnight cultures and growth curves in serial diluted 250mM Benzoate (microtitre plate). E. coli BW25113 benzoate/catechol MIC's. benE plasmid preps.
Ruby: Plasmid preparation of overnight cultures of transformed cells set up by supervisor Catherine. Digests to confirm the presence of target genes within the plasmids.
Darcy&Cate:OVernight cultures were preared which were then used for a colony PCR. The colony PCR was undertaken to identify sucesfully cloned plasmids. After unsucesfully identify any transformats, a digest and ligation experiment was repeated on benABCD from Rho9 and ADP1.
Luke; Performed overlap extension PCR following optimized protocol(Hilgarth and Lanigan, 2020). Of mCherry with CatM binding and BenM promoter with BenM.
Run on thyazoile orange gel.
Gel extracted mCherry with CatM binding(lane 2), BenM promoter with BenM(lane 3), and BenM promoter with GFP(lane 5).
Performed overlap extension PCR with 3500 whole construct and BenM promoter-GFP.
Run amplification of 3500 whole construct and BenM promoter-GFP, BenM-BenM binding, mCherry-CatM binding, mCherry-CatM binding(geneless) and GFP-BenM binding.photo
Run all amplifications on ethidium bromide gel,
BenM-BenM binding and mCherry-CatM binding had bands in correct positions(lane 2 and 3), although contained smear and other bands. All other overlap extension PCR failed, thus having light bands in incorrect positions.
Amplified CatM binding region(geneless) and BenM binding(geneless) from ADP1.
Performed overlap extension PCR of BenM-BenM binding with GFP and mCherry-CatM binding with CatM promoter.
Run on ethidium bromide gel BenM-BenM binding with GFP(lane 2), mCherry-CatM binding with CatM promoter(lane 3), CatM binding region(geneless)(lane 4) and BenM binding(geneless) from ADP1(lane 5), CatM binding region(geneless) with mCherry(lane 7) and BenM binding(geneless) with GFP(lane 8).
BenM-BenM binding with GFP, showed light band of correct size, although other bands were present.(lane 3)
mCherry-CatM binding with CatM promoter, contained band close to correct size, although contained large amount of smearing and other bands.(lane 4)
CatM binding region(geneless) and BenM binding(geneless) from ADP1, were unsuccessful with no DNA in lane 5 and 6.
mCherry and BenM binding(geneless) with GFP, were unsuccessful with no band in correct position.(lane 7 and 8)
Checked primer annealing temperature to observe if reason for other bands and smears within gels.
Created gradient PCR with all 9 separate fragments, 72 in total from 62°C
to 50°C.
Run on gel for 1 hour at 100 volts, with 2 sets of lanes on gel. All solutions run off gel and hit wells, thus unusable.
2nd try;
Created gradient PCR with all 9 separate fragments, 72 in total from 62°C
to 50°C.
run on gel at 100 volts for 20 minutes.
photo
Primer annealing for all primer much higher than expected
Each fragment is split between DNA ladder. Top left,section 1=fragment 1 from 60°C-50°C, then top middle, section2=fragment 2 from 60°C-50°C, then top right section 3=fragment 3 from 60°C-50°C, then bottom right section 4=fragment 4 from 60°C-50°C, then section 5 and 6.
Small gel. Top left section= section 7=fragment 7 from 72°C -60°C, then bottom right, section 8=fragment geneless BenM binding from 72°C -60°C, then right section starting from top then going to 2 on bottom right, section 9=fragment geneless catM binding from 72°C -60°C.
all lanes contained bands, except for fragment 7 which contained bands in 5 lowest temperatures(temp up to 66.1°C)
Performed a gradient overlap extension PCR for BenM-BenM binding with GFP and mCherry-CatM binding with CatM promoter. At temperatures 65, 63.5 58.9 and 57°C.
And amplified potential geneless fragment at temperatures 65, 63.5 58.9 and 57°C.
Band at the correct position for BenM-BenM binding-GFP, although other bands and large amount of smearing(lane 2-5).
No band but smearing across regions for mCherry-catM binding-CatM promoter and geneless construct(lane6-9).
Ruby: Digests of ADP1 transformations (cultures hadn't grown well enough to have plasmid preparations undertaken in previous week - Aakash undertook plasmid preparations on these cells. PCR reactions to stich together upstream, benE/benK and downstream gene fragments for gene knockouts.
Darcy&Cate: A colony PCR was undertaken on the transformants prepared in the previous week.
Luke;run ladder, BenM-BenM binding+GFP amplified fragment at 65, CatM, CatM binding+ Catm Promoter, small G(geneless construct), 40ul of each
Bands were not present in correct locations.
Aimed to furthy purify BenM-BenM binding and mCherry-CatM binding.
Placed in 2% agarose gel with thyazoile orange
running at 50 volts, 30 minutes, checked.
50 volts, 1 hour, checked.
70 volts, 30 minutes, checked.
30 minutes.
Then performed gel extraction
Then performed overlap extension PCR with BenM-BenM binding with GFP and mCherry-CatM binding with CatM promoter.
run 45ul of yesterdays reactions;
BenM-BenM binding with GFP and
mCherry-CatM binding with CatM promoter,
on thyazoile oranage gel.
100 volts for 1 hour.
Nothing within lanes, bp ladder present.
run PCR master mix of 5ul of reaction left over, amplyfiying sections
run on thyazoile orange gel.
Nothing within lanes, ladder present.
Aakash: E.coli knockout strains with inserted benE/benABCD clusters overnight cultures and growth curves in serial diluted 125mM Benzoate (microtitre plate). E. coli BW25113 (benE knockout strain) benzoate/catechol MIC's.
Ruby: Made calcium chloride competent E. coli BW 25113 cells (BenE knockout equivalent cells). Set up overnight cultures of WT E. coli 25113 on no selection media, Knockout E. coli cells in Kan and E. coli transformed with pTTQ18 plasmid in Amp. Performed transformations inserting plasmids containing Rhod 9 BenE, E. Ferg BenE, ADP1 benABCD and Rhod 9 benABCD into the competent cells.
Luke: from amplified mCherry-CatM binding performed restriction ligation using Ecor1 site and smar1 for blunt end, into PWH1266 plasmid. Transformed using ligation reactions, colonys present in control with no ligation reaction in them. tryed colony PCR and plasmid prep with restrictation digest. No plasmid with dedsired insert length was found.
obtained new primers that do not contain overlap regions.
Using 1ul solution containing CatM promoter-CatM gene and 1ul solution containing BenM binding-BenM gene(which contained many bands and large amount of smearing). Amplified these fragments using new primers and annealing temperature at 58°C, this resulted in less smearing and bands present within gel lane.
