Team:Athens/Notebook

• 
• Team
  Members Collaborations Attributions
• Project
  Description MERS-CoV Notebook Design Results Safety
• Wet Lab
  Experiments InterLab Parts
• Dry Lab
  Toehold Design Kinetic Modeling 3D Simulations Temperature Simulations
• Human Practices
  Overview Education & Public Engagement Integrated Human Practices
• Product
  Applied Design

Notebook

May

Getting familiarized with laboratory techniques and planning a timeline. Maria and Natalia will be primarily working at the Molecular Virology Laboratory of the Hellenic Pasteur Institute. Nelly, Elena and Panos will be mainly working at the Biotechnology Laboratory of NTUA and will be in charge of the Interlab study.

June

Hellenic Pasteur Institute

• Competent cells protocol (DH5a, BL21)
• Transformation and first trial with the Monarch® Plasmid Miniprep Kit | NEB using pGEM-luc as test plasmid
• Plasmid digestion using Xmnl, Xbal
• Getting familiar with agarose gel electrophoresis
• Gel extraction using Monarch® DNA Gel Extraction Kit | NEB
• Trehalase (TreA) isolation from BL21 E.coli cells
• DNA isolation using the QIAGEN® Plasmid Maxi Kit.

NTUA

• Interlab: followed the iGEM competent cells protocol, performed the competent cell test, with the kit provided by iGEM

July

Hellenic Pasteur Institute

• Design of primers for TreA, TreA-a, and TreA-b and selection of enzymes that will be used
• Design of leucine zippers for protein- protein interaction
• Digest of pET-15b and pGEM3zf(+) with NdeI, BamHI and EcoRI respectively
• Agarose gel electrophoresis for the pET-15b digest, gel extraction, Maxiprep of pET-15b using the QIAGEN® Plasmid Maxi Kit and transformation.

NTUA

• Unsuccessfully tried to transform competent cells with a GFP part found in the kit, aiming to use GFP as an alternative reporter protein for our system.
• While troubleshooting we tested the competence of the cells and concluded the error lied in the GFP plasmid.
• Interlab: Transformation with the plasmids provided by the iGEM Distribution Kit. OD and fluorescence measurements of our overnight cultures. Measurement of the fluorescence with Flow Cytometry. Followed the CFU protocol and counted colonies.

August

Hellenic Pasteur Institute

• PCRs using the Q5® High-Fidelity DNA Polymerase (M0491) | NEB and the Phusion® High-Fidelity DNA Polymerase | NEB
• PCR for TreA, phenol extraction of the pET-15b maxiprep DNA, double digestion of the pET-15b vector and the pure whole TreA using NdeI, BamHI and ligation of the pET-15b vector and TreA
• Transformation of pET-15b & TreA ligation product and minipreps. 1:10 ligation worked the best. Maxiprep of “plasmid A” (plasmid A= pET-15b vector + TreA insert). Acquisition of TreA expression plasmid.
• Purification of trehalase using the Protino® Ni-TED and Ni-IDA Packed Columns, in both native & denaturing conditions. Best results observed using the Ni-IDA column, in native conditions.
• PCRs for the TreA-a and TreA-b parts

September

Hellenic Pasteur Institute

• Elena joins the Pasteur lab team
• Polyacrylamide gel electrophoresis (10% (w/v) SDS PAGE) under reducing conditions, staining with coomassie blue.
• Benedict’s tests trials were performed using glucose solutions.
• Benedict’s, DNS tests were performed. DNS was proved to have greater sensitivity.
• Linearization of the maxi prepped pGEM vector via PCR, assembly of pGEM vector, Toehold Switches 3,7,8,16 and TreA-a,. Transformation. No colonies.
• Linearization PCR for triggers 3,7,8,16 vectors, PCR purification and assembly of triggers 3,7,8,16. Transformation of the digested products. No colonies.
• Assembly of zipper-b and TreA-b and transformation. No colonies.
• Tried gel extraction of the assembled product for all the unsuccessful transformations. No colonies.
• PCR cleanup kit on TreA whole, PCR linearization and deletion for Toehold switch 16 (TS plasmid + treA-a), confirmation PCR, assembly of TS and TreA. Transformation. No colonies.
• Again tried transformation after gel extraction of the assembled product and ethanol precipitation in both DH5a and XL1-Blue strains. Still no colonies.
• Tried transforming the gel extracted assembled vector after ethanol precipitation for every assembly product. Successful transformation in both DH5a and XL1-Blue strains (even though low number of colonies), for Toehold Switches 7,8,16, triggers 3, 16, zipper-b/ TreA-b. Colonies did not contain correct parts, except in the case of Toehold Switch 16.
• Plasmid DNA extraction and confirmatory digest (with PvuII for toehold switches, PvuII and EcorI for trigger RNAs, XbaI and SpeI for zipper-b/ TreA-b). Turns out that only Toehold Switch 16 and zipper-b/ TreA-b were successful.

October

Hellenic Pasteur Institute

• Nelly joins the Pasteur lab team.
• Digests of Toehold Switch 16 + TreA-a and igem vector using XbaI, SpeI. Dephosphorylation on igem vector using CIAP, to prevent self-ligation. Ligation on three different ratios. Transformation in both DH5a and XL1-Blue strains. No colonies.
• Transformation of the ligated product after gel extraction and ethanol precipitation. No colonies.
• Started the ligation process again. Still no colonies.
• At last, we obtained colonies from the ligation of part but didn’t have enough time to screen.
• Repeated successfully Trigger 16 assembly and transformation with freshly-made competent cells but there was not enough time to screen.
• Expression of TreA with the PURExpress® In Vitro Protein Synthesis Kit | NEB, in order to recreate the testing part. We used DNS and a glucometer to find out the time required for diagnosis.

Contact Info:

igemathens2018@gmail.com