Difference between revisions of "Team:UC San Diego/Notebook"

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<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Finished documentation for the enzymatic kinetics model
 +
<br>>Finished written documentation for constraint-based modeling
 +
<br>>Finished documenting COBRA scripts
 +
<br>>Set-up Github account for uploading scripts</p>
 
<span class="cd-date">WEEK 14</span>
 
<span class="cd-date">WEEK 14</span>
 
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<h2>Modeling</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Worked on documentation for the enzymatic kinetics model
 +
<br>>Began the development of a presentation in preparation for both the competition and upcoming talk at the library
 +
<br>>Finalized all figures and scripts.  
 +
<br><br>Finished tracing pathways for upregulation of light and growth in COBRA model
 +
<br>>Editing documentation, finding references
 +
<br>Making figures and tables for the presentation</p>
 
<span class="cd-date">WEEK 13</span>
 
<span class="cd-date">WEEK 13</span>
 
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<h2>Modeling</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Successfully managed to acquire results for the individual constructs under certain conditions.  
 +
<br>>Began documentation, finished searching for our references
 +
<br><br>Began documentation for constraint-based modeling
 +
<br><br>Started tracing pathways on reactions coupled with light and growth
 +
<br>>searched enzyme names on KEGG to identify upregulated subsystems
 +
</p>
 
<span class="cd-date">WEEK 12</span>
 
<span class="cd-date">WEEK 12</span>
 
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<h2>Modeling</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Reduced model as suggested by the work of D. Sekel and M. Iqbal in attempt to simulate in vivo behavior at steady state
 +
<br>>Began analysis of the different genetic constructs
 +
<br><br>Defined COBRA model in YPD media
 +
<br>>listed exchange reactions (reaction ids, reaction names) of both models in an excel document
 +
<br>>set bounds for inactive reactants to ‘0’
 +
<br>>less risk of noise and variation in fba.</p>
 
<span class="cd-date">WEEK 11</span>
 
<span class="cd-date">WEEK 11</span>
 
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<h2>Modeling</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Utilized model reduction strategies (i.e. quasi-steady state assumption, rapid equilibrium assumption, total quasi-steady state assumption etc.) in attempt to reduce our current model to one time-scale and cut the elapsed time for our simulations.  
 +
<br><br>Decided to focus on identifying pathways essential to light production
 +
<br>>started writing script to capture the fluxes through every reaction while varying growth and light
 +
<br>>Troubleshooting: only one fba solution for growth and light (linear relationship)</p>
 
<span class="cd-date">WEEK 10</span>
 
<span class="cd-date">WEEK 10</span>
 
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<h2>Modeling</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Finished code for the induction curve simulations and began generating preliminary results
 +
<br>>Explored light emission trace
 +
<br><br>Generated Robustness Analysis plots
 +
<br>>light vs. growth
 +
<br>>light vs. glucose
 +
<br><br>Generated Phenotype Phase Plane plots
 +
<br>>possible error in code, the graphs do not look as predicted
 +
<br><br>Updated both COBRA models with finalized reaction pathways</p>
 
<span class="cd-date">WEEK 9</span>
 
<span class="cd-date">WEEK 9</span>
 
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<h2>Modeling</h2>
 
<h2>Modeling</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Converted light production pathway and aldehyde synthesis pathway to a set of ODE equations
 +
<br>>Began simulations on MATLAB. In addition, most rate constants and fixed concentrations were found
 +
<br>>Checked the functionality of our model.
 +
<br><br>Completed the isolated COBRA model using the BiGG database
 +
<br>>unable to resolve layout of isolated model in CyFluxViz
 +
<br>>started working on the isolated model in the version 7 database</p>
 
<span class="cd-date">WEEK 8</span>
 
<span class="cd-date">WEEK 8</span>
 
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<h2>Modeling</h2>
 
<h2>Modeling</h2>
−
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit. Iusto, optio, dolorum provident rerum.</p>
+
<p>>Finished algorithm for parameter scan
 +
<br>>Finalized our core kinetic diagram
 +
<br>>Started search for rate constants</p>
 
<span class="cd-date">WEEK 7</span>
 
<span class="cd-date">WEEK 7</span>
 
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Revision as of 22:28, 18 September 2015

TIMELINE

September 24-28

Giant Jamboree!

Giant Jamboree!

September 16th

Library presentation at La Jolla Riford Library.

Read more! WEEK 14

Modeling

>Finished documentation for the enzymatic kinetics model
>Finished written documentation for constraint-based modeling
>Finished documenting COBRA scripts
>Set-up Github account for uploading scripts

WEEK 14

Wet Lab

+ Final preparations before the wiki freeze.

WEEK 14

Modeling

>Worked on documentation for the enzymatic kinetics model
>Began the development of a presentation in preparation for both the competition and upcoming talk at the library
>Finalized all figures and scripts.

Finished tracing pathways for upregulation of light and growth in COBRA model
>Editing documentation, finding references
Making figures and tables for the presentation

WEEK 13

Wet Lab

+ Mapped out errors in the parts that we assembled
+ Designed mutagenesis primers to fix recurring errors in C sequence

WEEK 13

September 4th

High school meet up.

Read more! WEEK 12

Modeling

>Successfully managed to acquire results for the individual constructs under certain conditions.
>Began documentation, finished searching for our references

Began documentation for constraint-based modeling

Started tracing pathways on reactions coupled with light and growth
>searched enzyme names on KEGG to identify upregulated subsystems

WEEK 12

Wet Lab

+ Added CDE fragments to AB plasmids via Gibson assembly
+ Designed Sanger sequencing primers
+ Miniprepped final plasmid clones

WEEK 12

Modeling

>Reduced model as suggested by the work of D. Sekel and M. Iqbal in attempt to simulate in vivo behavior at steady state
>Began analysis of the different genetic constructs

Defined COBRA model in YPD media
>listed exchange reactions (reaction ids, reaction names) of both models in an excel document
>set bounds for inactive reactants to ‘0’
>less risk of noise and variation in fba.

WEEK 11

Wet Lab

+ Attempted Gibson assembly of AB fragments
+ Miniprepped CDE fragments

WEEK 11

Modeling

>Utilized model reduction strategies (i.e. quasi-steady state assumption, rapid equilibrium assumption, total quasi-steady state assumption etc.) in attempt to reduce our current model to one time-scale and cut the elapsed time for our simulations.

Decided to focus on identifying pathways essential to light production
>started writing script to capture the fluxes through every reaction while varying growth and light
>Troubleshooting: only one fba solution for growth and light (linear relationship)

WEEK 10

Wet Lab

+ Analyzed sequencing data to identify error-free clones
+ Designed primers for PacBio Sequencing
+ Amplified error-free clones for all fragments

WEEK 10

Modeling

>Finished code for the induction curve simulations and began generating preliminary results
>Explored light emission trace

Generated Robustness Analysis plots
>light vs. growth
>light vs. glucose

Generated Phenotype Phase Plane plots
>possible error in code, the graphs do not look as predicted

Updated both COBRA models with finalized reaction pathways

WEEK 9

Wet Lab

+ Began subcloning of fragments into pUCGA vector to find error-free clones

WEEK 9

August 6th

Southern California iGEM Meetup

Read more! Week 8

Modeling

>Converted light production pathway and aldehyde synthesis pathway to a set of ODE equations
>Began simulations on MATLAB. In addition, most rate constants and fixed concentrations were found
>Checked the functionality of our model.

Completed the isolated COBRA model using the BiGG database
>unable to resolve layout of isolated model in CyFluxViz
>started working on the isolated model in the version 7 database

WEEK 8

Wet Lab

+ Received our DNA fragments from SGI thanks to the BioXP!
+ Attempted amplify fragments via PCR for assembly and transformation.

WEEK 8

Modeling

>Finished algorithm for parameter scan
>Finalized our core kinetic diagram
>Started search for rate constants

WEEK 7

Wet Lab

+ Assembled Interlab Devices. Resuspended and measured them successfully.
+ Prepared YPD plates.

WEEK 7

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

WEEK 6

Wet Lab

+ Continued interlab study with miniprep, restriction digests, ligation, gel electrophoresis and gel purification.

WEEK 6

Modeling

>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

WEEK 5

Wet Lab

+ Started interlab study by transforming parts from the 2015 distribution.

WEEK 5

Modeling

>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

WEEK 4

Wet Lab

+ Sent our genes to SGI and ordered lab supplies. Almost ready to go!

WEEK 4

Modeling

>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)

WEEK 3

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.

WEEK 3

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

WEEK 2

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.

WEEK 2

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

WEEK 1

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.

WEEK 1