Team:UFRGS Brazil/LabBook
Voltammetry tests for glyphosate dosage - 04/12/2019
Electrolytic cell assembly
Special \(20 mL\) becker, special de \(20 mL\), Potentiostat/Galvanostat Buffer (generate V), auxiliary electrode (platine), reference (Ag/AgNO3)
Glassy carbon electrode assay
Cell: red wire test, referential green, blue auxiliary electrode.
Solution: \(20 mL\) of H20 milliQ, Phosphate Buffer pH = 7 \(2 mL\), \({Cu}^{2+}\) 500 \(mg/mL\) 20 \(\mu L\)
Glyphosate added at \(20 \mu L\) starting from \(34 mM\) solution.
No analytical result
Test 2 - 04/24/19
Electrolytic cell assembly
Trying another kinds of electrode: copper (wire), gold, carbon ink (3d printed), carbon nanotubes
Same Cell
No analytical result
Test 3 - 04/30/19
Addition of counter-ion on cells: KCL
Tests iterations
Test 4 - 05/03/19
Change of buffers: ammonium chloride pH: 10 and sodium acetate pH: 4
No results
Test 5 - 05/09/19
Returning to glassy carbon electrode
New copper solution
Test 6 - 05/16/19
New glyphosate solution (may be easily degraded)
New copper solution
Test 7 - 05/23/19
Defining new strategies
Test 8 - 06/06/19
Deaerate solutions
No analytical results
Move on to HPLC strategies
Construction of Promoter Cassettes - 06/06/19
Firstly, the promoters pKat and pJ23100 were chosen. Both came as ssDNA from synthesis. They were mixed together and heated to make dsDNA. The resistance cassette (CmR_Cas) came from synthesis as well, but as dsDNA.
07/06/19
The CmR_Cas PCR was carried out again with 35 cycles. Fragment size was confirmed by agarose gel. CmR_Cas was purifies using PureLink purification Kit (Invitrogen).
Generation of homologous flanking sequences Resistance + Promoter
PCR was carried out to add flanking sequences in pKat and pJ23100 homologous to CmR_Cas. The same was done to CmR_Cas, to add homologous sequences to each of the promoters. CmR PCR was carried out as recommended by the manufacturer instructions (12 cycles). There were no bands visible in agarose gel.
09/06/19 - Single-Joint PCR – Fusion Resistance + Promoter
Single-joint PCR (primerless PCR) was done to fuse CmR_Cas with each promoter. The product of this PCR was diluted and used as template to a second round of PCR. Product was migrated in agarose gel, but bands were in the wrong size. Melting temperature (Tm) was thought to be the problem. The same PCR was done, with 5 different Tms (58, 60, 64, 68, 70 ℃) for both promoters. All PCR products were migrated in agarose gel, but all showed the same band size. Tm was not the problem.
11/06/19 - Generation of homologous flanking sequences Resistance + Promoter (2)
With the arrival of Q5 polymerase, we decided to restart all amplifications. Now, pLac was added to the promoters. All three promotores were PCR amplified to add homologous flanking sequences as described previously. The same was carried out to add flanking sequences to CmR_Cas homologous to each promoter (pKat, pJ23100 and pLac). PCR products of promoters (~100 – 250 bp) were confirmed and purifies in TEB 3% agarose gel. PCR products from CmR_Cas amplification were confirmed and purified from TAE 1% agarose gel.
12/06/19 – Single-Joint PCR - Fusion Resistance + Promoter (2)
Single-joint PCR (primerless PCR) was done to fuse CmR_Cas with each promoter. The product of this PCR was diluted and used as template to a second round of PCR. The three final cassettes were size confirmed and purified from agarose gel using PureLink purification Kit (Invitrogen)
01/07/19
Pre-culture of Escherichia coli DH5\(\alpha\) containing PKD46 (lamba-Red recombinase plasmid) in LB medium + ampicillin
02/07/19
Miniprepreparation of PKD46 plasmid.
03/07/19
Plasmid confirmation was done by gel electrophoresis, and quantification by qubit.
04/07/19
E. coli K12 MG1655 was transformed via electroporation to add PKD46 plasmid (room temperature protocol)
08/07/19
Pre-culture of E. coli K12 harboring PKD46.
09/07/19
Minipreparation of PKD46. Confirmation was done by gel electrophoresis. Glycerol stocks were made and stored at -80℃.
10/07/19
Pre-culture of E. coli K12 harboring PKD46 on LB medium + ampicillin.
11/07/19
Transformation of E. coli K12 with the three promoter cassettes (pKat, pJ23100, pLac). Chloramphenicol was used as selective agent.
12/07/19
Colonies were too small for PCR. Colonies were plated on new LB + chloramphenicol plates.
15/07/19
Colonies were confirmed by DNA extraction and PCR. Glycerol stocks were made and stored at -80℃
22/07/19
Preparation of IDT primers. Design of new primers.
23/07/19
Pre-culture of mutants harboring promoter cassettes (k1, k2, j1, j5, L8, L16) in LB + chloramphenicol.
24/07/19
Transformation by electroporation of PKD46 in mutants k1, k2, j1, j5, L8, L16
29/07/19
Pre-culture of mutants k1, k2, j1, j5, L8, L16 harboring PKD46 in LB + chloramphenicol + ampicillin.
30/07/19
Minipreparation of PKD46 from mutants k1, k2, j1, j5, L8, L16
31/07/19
Plasmid confirmation was done by gel electrophoresis.
20/08/19
Pre-culture of mutants harboring promoter cassettes (k1, k2, j1, j5, L8, L16) in LB medium with chloramphenicol. Pre-culture of E. coli K-12 in LB medium.
22/08/19
Growth curve was carried out in M9 and M9 with 1/5 of phosphate in a 96-well plate.
23/08/19
24h point of growth curve.
26/08/19
Sodium alginate was diluted in different concentrations. CaCl 4% was prepared.
Pre-culture of E. coli K12 in LB medium.
27/08/19
Expanded pre-culture to 200 ml medium.
28/08/19
Culture was centrifuged and resuspended in different concentrations of alginate. Three different sized spheres were made for each of the 4 alginate concentrations. Spheres were collected in different times (15 min, 30 min, 1h and 24h).
02/09/19
PCR was carried out to add homologous flanking sequences to phnEG_Cas (70 nt chimeric primer). Did not work.
03/09/19
Different annealing temperatures were tested to add homologous flanking sequences to phnEG_Cas. Did not work.
09/09/19
TouchDown-PCR (TD-PCR) was carried out to try to correct amplification issues with the chimeric primers of phnEG_Cas, with two different annealing times. Did not work.
10/09/19
phnEG_Cas was PCR amplified with the Forward chimeric primer and reverse conventional primer (generating phnEG_FC). phnEG_Cas was PCR amplified with the forward conventional primer and reverse chimeric primer (generating phnEG_RC).
11/09/19
Fragments phnEG_FC and phnEG_RC were size confirmed by gel electrophoresis and purified from agarose gel. Both fragments were mixed at 1:1 molar ratios and used as template to PCR, with the chimeric 70 nt primers. Did not work.
12/09/19
Pre-culture of mutants harboring the promoter cassette and PKD46 plasmid in LB + cloranfenicol + ampicilina.
13/09/19
Fragments phnEG_FC and phnEG_RC were mixed in 1:1 molar ratio and heated to 95 ℃ for 5 minutes. After cooling, the product was used to transform mutants harboring the promoter cassette and PKD46 plasmid by lambda-red recombinase electroporation protocol. Transformation was plated on LB + chloramphenicol plates, in 25, 50, 100 and 150 \(\mu L\).
16/09/19
Transformation produced too many colonies, even in the 25 \(\mu L\) plate as the antibiotic resistance was the same from before transformation. Colonies from the 25 \(\mu L\) plate were streak-plated on M9 medium with \(1\over 5\) phosphate + chloramphenicol + (50 ppb or 500 ppb) of glyphosate, in order to produce single colonies with an alternative selection method. E. coli K12 was streak-plated as well, in order to see the phenotype.
17/09/19
Colony PCR was done with 60 single colonies (20 for each promoter) from the streak-plate on M9 medium. PCR product was migrated on TAE agarose gel, with no positive colonies.
18/09/19
Literature review for alternative media to be used as selective media, with glyphosate as the sole source of phosphate. Found Tris-Glucose (TG) medium.
19/09/19
Streak-plated colonies from the 25 \(\mu L\) transformation plate in TG medium + chloramphenicol + (500 ppb or 50 ppm) of glyphosate.
20/09/19
Colony PCR was done with 60 single colonies (20 for each promoter) from the streak-plate on TG medium. Found a few with putatively positive results.
23/09/19
pJ23100 colonies did not show positive results. Pre-culture of three colonies from pKat, three colonies from pLac and WT in LB medium. PCR positive colonies for locus-insertion confirmation.
24/09/19
Growth curve assay was carried out with pre-cultured bacteria, in technical triplicate, and biological duplicate. \({OD}_{600}\) was initially standardized to 0.05. Growth points were measured at 0h, 1h, 2h, 3h, 4h, 5h and 6h.
25/09/19
24 hour growth-curve point. Pre-culture E. coli K12 in LB medium.
26/09/19
Preparation of chemically competent K12 cells.
10/10/19
DNA sequences (finally) arrived from regulatory agents.
Fragments were named as follow:
Fragment 1: BBa_K259007 - AraC Promoter fused with RBS
Fragment 2: cI Repressor Cassette (AraC promoter fused with RBS + cI CDS + Terminator + cI Repressed promoter)
Fragment 3: lacI Protein generator (Promoter + RBS + lacI CDS + Terminator)
Fragment 4: lacI induced repressor cassette (lacI regulated promoter + RBS + cI CDS + Terminator + cI repressed promoter)
Fragment 5: improved lacI induced repressor cassette (lacI lambda pL Hybrid regulated promoter + RBS + cI CDS + Terminator + cI repressed promoter)
Fragment 6: MazF translation unit fused with a terminator (RBS + CDS + Terminator)
Fragment 7: mRFP translation unit fused with a terminator (RBS + CDS + Terminator)
Fragment 8: Tse2 translation unit fused with a terminator (RBS + CDS + Terminator)
All fragments (1-8) and plasmids pSB1C3, pSB1A3 and pSB1K3 were cut with EcoRI and PstI, in order to generate stocks.
Fragment 1 was cut, in a different reaction, with EcoRI and SpeI.
Fragment 2 was cut, in a different reaction, with EcoRI and SpeI
Fragment 7 was cut, in a different reaction, with XbaI and PstI.
Fragment 8 was cut, in a different reaction, with XbaI and PstI.
All cleavage reactions were kept at 37 oC overnight.
11/10/19
Fragments 1, 2, 3 and 5 were fused to pSB1C3, in order to generate stocks.
Fragments 4, 6, 7 and 8 were fused to pSB1A3, in order to generate stocks.
Fragment 1 (EcoRI/SpeI) was fused to fragment 7 (XbaI/PstI), in order to generate composite part BBa_K3215011.
Fragment 2 (EcoRI/SpeI) was fused to fragment 7 (XbaI/PstI), in order to generate composite part BBa_K3215012.
Fragment 2 (EcoRI/SpeI) was fused to fragment 8 (XbaI/PstI), in order to generate composite part BBa_K3215013.
All fusion reactions were kept overnight at room temperature (around 23 oC). Enzyme T4 DNA ligase (Promega) was used in all reactions.
12/10/19
Individual sequences (1-8), plus composite parts 1.7, 2.7 and 2.8 were migrated on agarose gel for ligation size confirmation. Ligation was confirmed in all 11.
All individual sequences (1-8) were transformed by heat shock in chemically competent E. coli K12 cells, and plated on LB + the appropriate antibiotic (chloramphenicol or ampicillin).
Composite parts BBa_K3215011, BBa_K3215012 and BBa_K3215013 were individually ligated with pSB1K3. Reactions were kept overnight.
13/10/19
Composite parts BBa_K3215011, BBa_K3215012 and BBa_K3215013 were individually transformed by heat shock in chemically competent E. coli K12 cells, and plated on LB + kanamycin, with the exception of BBa_K3215013, that was also plated with 0.2% arabinose, due to kill-switch system.
14/10/19
No colonies for BBa_K3215013. Colony PCR was done to confirm BBa_K3215011 and BBa_K3215012. Product was migrated on Agarose gel. BBa_K3215011 showed positive colonies, BBa_K3215012 did not.
Fragments 2, 7 and 8 were cut again, with EcoRI and SpeI for fragment 2, and XbaI and PstI for fragments 7 and 8. Enzyme loads were increased. Reactions were kept overnight.
17/10/19
Fragment 2 (EcoRI/SpeI) was fused to fragment 7 (XbaI/PstI), in order to generate composite part BBa_K3215012.
Fragment 2 (EcoRI/SpeI) was fused to fragment 8 (XbaI/PstI), in order to generate composite part BBa_K3215013.
All fusion reactions were kept overnight at room temperature (around 23 oC). Enzyme T4 DNA ligase (Promega) was used in all reactions.
18/10/19
Composite parts BBa_K3215012 and BBa_K3215013 were individually transformed by heat shock in chemically competent E. coli K12 cells, and plated on LB + kanamycin (with 0.2% arabinose for BBa_K3215013).
19/10/19
No colonies were obtained from BBa_K3215013. Colony PCR was done with BBa_K3215012.
Pre-culture of positive colonies of BBa_K3215011 and BBa_K3215012 in LB + kanamycin, and E. coli K12 WT in pure LB.
20/10/19
Pre-cultured bacteria were inoculated in a 96 deep-well plate, in technical triplicate and biological duplicate with LB, LB + 0.01, 0.05, 0.1, 0.2 and 1% of arabinose. Chloramphenicol was added to all wells, with exception of WT wells. RFP fluorescence points were measured at 0, 30 min, 1h, 2h and 3h.
Plasmid miniprepation was done with the same pre-inoculum, by alkaline lysis.
Plasmids were cut with EcoRI and PstI for size confirmation, and kept overnight at 37 oC.
E. coli harboring BBa_K3215011 and BBa_K3215012 were also plated on LB + chloramphenicol with different concentrations of arabinose, in order to see fluorescence in the microscope.
21/10/19
Colonies from the second transformation of BBa_K3215012 and BBa_K3215013 were inoculated in 1 mL of LB + chloramphenicol, and LB + chloramphenicol with 0.5% of arabinose, and were kept at the thermomixer, at 37 ℃, for 7 hours. RFP fluorescence was measured for BBa_K3215012, and \({OD}_{600}\) was measured for BBa_K3215013.
PCR was done with primers VF2 and VR using 2 microliters of the 1 mL inoculum, to confirm colonies. Product was migrated on agarose gel.
