Team:UFRGS Brazil/Notebook

iGEM UFRGS
Who are we?

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 2mL, Cu2+ 500 mg/mL 20 uL

-Glyphosate added at 20 uL starting from 34 mM solution.

-No analytical result

Voltammetry test 2 - 04/24/19

-Trying another kinds of electrode: copper (wire), gold, carbon ink (3d printed), carbon nanotubes

-Same Cell

-No analytical result

Voltammetry test 3 - 04/30/19

-Addition of counter-ion on cells: KCL

-Tests iterations

Voltammetry test 4 - 05/03/19

-Change of buffers: ammonium chloride pH: 10 and sodium acetate pH: 4

-No results

Voltammetry test 5 - 05/09/19

-Returning to glassy carbon electrode

-New copper solution

Voltammetry test 6 - 05/16/19

-New glyphosate solution (may be easily degraded)

Voltammetry test 7 - 05/23/19

-Defining new strategies

Voltammetry 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 aswell.

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.

06/07/19

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).

06/09/19 - 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 oC) for both promoters. All PCR products were migrated in agarose gel, but all showed the same band size. Tm was not the problem.

06/11/19

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.

06/12/19 – 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)

07/01/19

-Pre-culture of Escherichia coli DH5α containing PKD46 (lamba-Red recombinase plasmid) in LB medium + ampicilin

07/02/19

-Miniprepreparation of PKD46 plasmid.

07/03/19

-Plasmid confirmation was done by gel eletrophoresis, and quantification by qubit.

07/04/19

-E. coli K12 MG1655 was transformed via electroporation to add PKD46 plasmid (room temperature protocol).

07/08/19

-Pre-culture of E. coli K12 harboring PKD46.

07/09/19

-Minipreparation of PKD46. Confirmation was done by gel electrophoresis.

-Glycerol stocks were made and stored at -80C.

07/10/19

-Pre-culture of E. coli K12 harboring PKD46 on LB medium + ampicilin.

07/11/19

-Transformation of E. coli K12 with the three promoter cassettes (pKat, pJ23100, pLac). Chloranfenicol was used as selective agent.

07/12/19

-Colonies were too small for PCR. Colonies were plated on new LB + chloranfenicol plates.

07/15/19

-Colonies were confirmed by DNA extraction and PCR.

-Glycerol stocks were made and stored at -80C

07/22/19

-Preparation of IDT primers. Design of new primers.

07/23/19

-Pre-culture of mutants harboring promoter cassettes (k1, k2, j1, j5, L8, L16) in LB + chloranfenicol.

07/24/19

-Transformation by electroporation of PKD46 in mutants k1, k2, j1, j5, L8, L16.

07/29/19

-Pre-culture of mutants k1, k2, j1, j5, L8, L16 harboring PKD46 in LB + chloranfenicol + ampicilin.

07/30/19

-Minipreparation of PKD46 from mutants k1, k2, j1, j5, L8, L16.

07/31/19

-Plasmid confirmation was done by gel electrophoresis.

08/20/19

-Pre-culture of mutants harboring promoter cassettes (k1, k2, j1, j5, L8, L16) in LB medium with chloranfenicol.

-Pre-culture of e. Coli k-12 in LB medium.

08/22/19

-Growth curve was carried out in M9 and M9 with ½ of phosphate in a 96-well plate

08/23/19

-24h point of growth curve.

08/26/19

-Sodium alginate was diluted in different concentrations. CaCl 4% was prepared.

-Pre-culture of E. coli K12 in LB medium.

08/27/19

-Expanded pre-culture to 200ml medium.

08/28/19

-Culture was centrifuged and ressuspended 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).

09/02/19

-PCR was carried out to add homologous flanking sequences to phnEG_Cas (70 nt chimeric primer). Did not work.

09/03/19

-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.

09/10/19

-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).

09/11/19

-Fragments phnEG_FC and phnEG_RC were size confirmed by gel electrophoresis and purified from agarose gel. Both fragments were mixed and used as template to PCR, with the chimeric 70 nt primers. Did not work.

09/12/19

-Pre-culture of mutants harboring the promoter cassette and PKD46 plasmid in LB + chloranfenicol + ampicilin.

09/12/19

-Fragments phnEG_FC and phnEG_RC were mixed together in 1:1 molar ratio and heated to 95C for 5 minutes. After cooling, the product were used to transform, by electroporation, mutants harboring the promoter cassette and PKD46 plasmid.

Sponsors

La Naturelle
Laura Grisci
Fermmento Labs
Hidrosul
Fornarolli
Fornarolli