Team:UC San Diego/Notebook
TIMELINE
September 24-28
Giant Jamboree!
Giant Jamboree!
Modeling
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WEEK 14Wet Lab
+Final preparations before the wiki freeze.
WEEK 14Modeling
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WEEK 13Wet Lab
+ Mapped out errors in the parts that we assembled
+ Mutagenesis to fix recurring errors in C sequence
Modeling
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WEEK 12Wet Lab
+ Attempted Gibson Assembly with CDE with AB
+ Designed sequence primers
+ Miniprepped full size clones
Modeling
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WEEK 11Wet Lab
+ Attempted full gibson assembly of AB fragment
+ Miniprepped CDE fragments
Modeling
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WEEK 10Wet Lab
+ Selected error free clones for CDE fragments from sequencing data
+ Designed primers for PacBio Sequencing
+ Transformed error free clones of CDE
Modeling
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WEEK 9Wet Lab
+ Assembled using Gibson Assembly and sequenced CDE fragments
+ PCR and transformed fragments
Modeling
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WEEK 8Wet Lab
+ Received our DNA fragments from SGI thanks to the BioXP!
+ Attempted amplify fragments via PCR for assembly and transformation.
Modeling
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WEEK 7Wet Lab
+ Assembled Interlab Devices. Resuspended and measured them successfully.
+ Prepared YPD plates.
Modeling
>Updated core model to include intermediate species, such as the primary light emitter, and other key reactions, such as aldehyde inhibition, that are key for continuous light emission
Researching literature for useful COBRA model constraints
>made a table of uptake and secretion fluxes in units of mmol/(g*hr)
>found a paper, Biomass composition: the ‘‘elephant in the room’’ of metabolic modelling
>contacted professor for supplemental information
Wet Lab
+ Continued interlab study with miniprep, restriction digests, ligation, gel electrophoresis and gel purification.
WEEK 6Modeling
>Literature research
>Looking for techniques to combine the deterministic model and genome-scale model
>Attended the Q-bio Summer School: Computational Synthetic Biology
Learned to code SBML
>added reactions and metabolites manually to version 7 model since the built-in function addReactions was unusable
Wet Lab
+ Started interlab study by transforming parts from the 2015 distribution.
WEEK 5Modeling
>Searched for genome-scale model parameters
>Developed core network diagram and found some nominal parameters for ODE model
>Constructed core model for our bioluminescent system
>Compile various literature sources necessary to understand bioluminescent network
>Developed kinetic equations corresponding to network
Thinking of making an isolated COBRA model to visualize flux distribution
>installed Cytoscape and CyFluxViz
>investigation on exporting model and fluxes from Matlab structure format
Wet Lab
+ Sent our genes to SGI and ordered lab supplies. Almost ready to go!
WEEK 4Modeling
>Developed lecture on enzymatic kinetics and gene expression modeling, along with corresponding MATLAB simulations
Continued Investigation of literature on lux system reactions in-detail
>attempted to link pathways to rate-constants found in literature
>made first digitalized diagram of the entire bioluminescent system
Installed MATLAB student copy sponsored by iGEM
> Made first script to add igem reactions
Installed APE
> thinking about incorporating protein synthesis into the COBRA model
Investigation about the CrabTree Effect in s288c
>could affect how much light is made (acetate is a substrate)
Wet Lab
+ Added frp gene sequence from Vibrio Harveyi to our plasmid to stabilize luminescent output.
+ Codon optimized our plasmid sequences for expression in yeast.
Modeling
Became familiarized with genome-scale modeling and metabolic control analysis
Literature research: Understanding the different components of the bioluminescent system
>aldehyde synthesis
>light production
Researched Literature on COBRA analysis and methods
>used e.coli as a toy model
>ran stimulations of FBA, robustness analysis, and phenotype phase plane
Wet Lab
+ Created a preliminary design for the plasmids using ApE.
+ Improved them over the week by adding tags, removed illegal restriction sites, and changing repetitive sequences.
Modeling
Developed and finalized Primer to MATLAB Programming
Researched potential modeling avenues
>genome scale analysis of synthetic construct
>analysis of the tradeoffs between different genetic circuit designs and gene dynamics
>analysis of metabolic activity in an isolated network via Metabolic Control Analysis
Browsed recent literature for lux system
>identified its advantages and disadvantages as a reporter system
>differences in luciferase systems of beetles and bacteria
Familiarized ourselves with basics of enzyme kinetics
>Michaelis-Menten equations
Downloaded COBRA Toolbox plugin for Matlab
Wet Lab
+ Found genes coding for fatty acid reductase complex that have been validated in an in vitro synthesis paper.
+ Planned how to assemble our plasmids and determined nucleotide sequences for lux A-E of Photobacterium Phosphoreum.
+ Compared the amino acid sequences to those of other organisms on BLAST to check for significant discrepancies in our sequence.