Team:Aberdeen Scotland/Notebook/Summer

From 2014.igem.org

Team:Aberdeen Scotland/Notebook - 2014.ogem.org



Project Timeline

Responsibilities and activities during the course of the project over the summer.


Winning Project Idea

E. coli quorum sensing based system for detecting co-localized disease markers for Trypanosomiasis Diagnostic Set

  • Our lab activities were split into four major streams focusing on:
    • Ag43
    • INP
    • Quorum sensing
    • Detector device construction and mathematical modelling

  • Week 1 23/6-29/6
    • Introduction to the lab and familiarization with safety rules
    • Project planning
    • Preparation of stock solutions and other materials
    • Rescuing of desired BioBricks
    • Initial plasmid mini preps
    • Standard restriction digest of rescued BioBricks and gel electrophoresis
    • Design of sequencing primers for two desired BioBricks (Bba_K759001 and BBa_K523013)
    • Contacting local media in order to promote synthetic biology, iGEM and our project
    • Meeting with a specialist in the field of antibodies to discuss out project idea
    • Identification of relevant parts of the biological system for modelling
    • Preliminary modelling decisions (modelling software, approach, etc)

  • Week 2 30/6-6/7
    • Trial PCR
    • E. Coli growth curve assemby
    • Further characterization and write-up of three BioBricks we found not to be compatible with RFC10 assembly standard (Bba_K759001, Bba_K542009, Bba_K346007)
    • Primer design for multiple site-directed mutagenesis to remove 6 additional PstI sites present in Bba_K759001
    • Primer design for FLAG+MCS insertion into Ag43 and INP-YFP via in-fusion
    • Primer design for HIS+MCS insertion into Ag43 and INP-YFP via in-fusion
    • Primer design for beta-hairpin removal from Ag43 via in-fusion to remove aggregation properties
    • Transformations of plasmids of all rescued BioBricks into competent cells (XL-1 Blue); plating
    • Preparation of application to WHO, in order to request blood and serum samples from HAT sufferers at different stages of disease -> could not work with samples due to Health and Safety concerns :(
    • Friday lab meeting to discuss project progress and evaluate aims of the project
    • Started work on spatial model of Quorum Sensing in order to decide on most appropriate cell-to-cell communication design

  • Week 3 7/7-13/7
    • Site-directed mutagenesis; two experimental conditions: with 3 pairs of primers and all 6 pairs of primers (PCR, DpnI digest, transformations into XL-1 Blue competent cells, plating, choosing clones for further analysis (24), overnight cultures, plasmid mini-preps, restriction digest with PstI; data analysis)
    • Further analysis of clones with XbaI+SpeI digest
    • Selection of two candidates with the least number of PstI sites remaining for the second round of SDM
    • iGEM track specification
    • Friday lab meeting to discuss project progress and evaluate aims of the project
    • Preliminary results of Quorum Sensing model in 2D (bad news for original design)
    • Started work on initial web design for Wiki

  • Week 4 14/7-20/7
    • Sending selected candidates from SDM no.1 for sequencing
    • SDM attempt no.2 with appropriate primers to remove remaining PstI sites from selected candidates (PCR, DpnI digest, transformations into XL-1 Blue competent cells, plating -> no colonies :()
    • Re-transformations -> no success :(
    • group pictures taken for promotional materials
    • Friday lab meeting to discuss project progress and evaluate aims of the project
    • Further analysis of Quorum Sensing by 3D modelling (initial QS design needs to be adjusted)
    • Restructured Wiki design for optimization

  • Week 5 21/7-27/7
    • Investigation into artificial gene synthesis in order to replace part of the gene with remaining PstI sites
    • Repetition of SDM no.2 with increased template concentration and a temperature gradient; DMSO added to one condition
    • DpnI digest, transformations into XL-1 Blue competent cells, plating, choosing clones for further analysis (50!), overnight cultures, plasmid mini- preps, restriction digest with PstI; data analysis)
    • Potential candidates with all additional PstI sites removed were selected for full restriction digest panel
    • Optimization of gel electrophoresis to achieve desired resolution
    • Graphic design of the project overview figure
    • Friday lab meeting to discuss project progress and evaluate aims of the project
    • Multi-cellular simulations of Quorum Sensing confirming the correction and improvement of cell-to-cell communication design
    • Started work on GFP production model
    • More work on Wiki design

  • Week 6 28/7-3/8
    • Restriction digest with unique cutters to investigate band pattern in order to detect whether significant insertions or deletions took place
    • Sending two potential candidates lacking all undesired PstI cut sites off for sequencing
    • In-fusion to insert FLAG flanked by multiple cloning sites into Ag43 PstI-free candidate; simultaneous insertion into ampicillin and chloromphenicol backbones (gradient PCR, DpnI digest, gel electrophoresis, restriction digest of backbones+RFP constructs and gel extraction, PCR clean-up, electrophoresis, in-fusion, transformation, plating, overnight cultures, candidate selection)
    • Diagnostic restriction digest with XbaI+HindIII and BglII+SpeI to detect presence of inserted MCS
    • One Ag43+FLAG+MCS candidate on ampicillin backbone selected; we didn't achieve corresponding clone on chloromphenicol backbone
    • Meeting with the artist to discuss logo project and public outreach-related images
    • Friday lab meeting to discuss project progress and evaluate aims of the project
    • Investigation of GPF production model - all good, no surprises
    • Started work on antibody binding model
    • Populated Wiki with preliminary info
    • Created TEXT to DNA Converter and added to Wiki

  • Week 7 4/8-10/8
    • Ag43+FLAG+MCS candidate on ampicillin backbone sent off for full-sequencing
    • Induction of the construct with arabinose
    • Western Blot to investigate expression of FLAG tag
    • In-fusion to remove beta-hairpins from Ag43+FLAG+MCS construct; two attempts: PCR optimization
    • Preparation for in-fusion to remove beta-hairpins attempt no.3 (PCR, DpnI digest, gel electrophoresis)
    • Friday lab meeting to discuss project progress and evaluate aims of the project
    • Antibody binding model investigation - interesting results
    • Bringing the model together and confirming the design of the system

  • Week 8 11/8-17/8
    • In-fusion on prepared PCR fragments (in-fusion, transformation into DH5α competent cells, plating, overnight cultures (small), plasmid mini-preps)
    • Restriction digest analysis with KpnI+EcoRI -> no bands visible on the gel; nanodrop confirmed very low concentrations of obtained plasmid
    • Repetition of plasmid mini-preps; the same low yield recorded
    • Abstract and project overview write-up for iGEM submission
    • Submission of the first article for publishing in AU Science magazine
    • VISA interview in London! :)
    • Contacted Physics Department to acquire components for construction of fluorescence detector
    • Finalizes TEXT to DNA converter inner workings (Wiki)
    • Design cleanup and more info added to Wiki

  • Week 9 18/8-24/8
    • Tuesday lab meeting to discuss project progress and evaluate aims of the project
    • Re-transformation of in-fusion products into different competent cells (Stellar supercompetent cells); plasmid mini-prep yielded much higher plasmid concentrations
    • Diagnostic restriction digest with XbaI+KpnI and HindIII+PstI; candidate with removed beta-hairpins chosen
    • Candidate sent off for sequencing
    • Backbone swap of Ag43+FLAG+MCS (ampicillin) into chloromphenicol backbone for submission; full restriction enzyme panel
    • Co-transformations of Ag43+FLAG+MCS (amp) with a GFP Producer Controlled by 3OC6HSL Receiver Device (cm) (Bba_T9002) to create a reporter construct for Quorum Sensing analysis
    • Friday lab meeting to discuss project progress and evaluate aims of the project
    • Fluorescence detector design completion and initial tests
    • Populate Wiki with project details and expanded design capabilities to accommodate final version

  • Week 10 25/8-31/8
    • Ag43+FLAG+MCS (-)beta hairpins construct transferred onto chloromphenicol backbone for submission; diagnostic full restriction enzyme panel done
    • Induction of all Ag43 constructs with arabinose; aggregation assay
    • Western Blot of Ag43+FLAG+MCS (-)beta hairpins to test expression of FLAG
    • Surface expression test on all created constructs expressing FLAG using magnetic beads coated with anti-FLAG pull down
    • Preparation of all created BioBricks for iGEM submission; registry of parts numbers assigned
    • Preparation for the PekaKucha Explorathon presentation, European Researchers Night
    • Submission of the second article for publishing in AU Science magazine
    • Final lab meeting to summarize achievements and discuss preparation of presentation and poster for Giant Jamboree in Boston - champagne included! :) :)
    • Final tests of fluorescence detector

  • Subsequent accomplishments
    • Third article for AU Science magazine
    • PekaKucha Explorathon presentation at the European Researchers Night


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