IGEM:MIT/2006/Notebook/2006-8-14

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Contents

To do

  1. nanodrop and sequence 32 minipreps (yes that is 64)
  2. look at incoming sequence order
    • throw out all duplicate (A, B, C, etc.) glycerols and minipreps if all the intermediate parts we sequenced look good
  3. smell the EZ media smellers and set-up smell test II (andre)
  4. plate reader results? plan for osmY?? repeat failed 3-part assembly to E0840?
  5. hook pCHBA up to terminator
  6. digest RBS.BAT2s with E and S, check gel, if correct, hook up to terminator
  7. digest THIs with X and P, check gel, if correct, hook up to RBS
  8. made LC of R0011 to try to hook up to RBS.coding.terms

Preliminary Smell Test II (André)

Experiment description

  • We would like to know the amount of cells (and, therefore by correlation, roughly the amount of protein) necessary to produce the smells

Proposed Experimental Design

  • Add appropriate amounts of precursors to media with roughly equivalent numbers of cells
  • Smell cultures at different time intervals
  • Read OD at different time intervals.

We hope to generate data that resemble the following:

Cellular Entry of Precursor: Effects of pH

Experiment Motivation

  • Transmembrane transport is a ______ process. Few small molecules are able to freely diffuse across the lipid bilayer. Many molecules only gain entry to the cell with the help of transporter proteins and ATP. Because we are adding the precursor exogenously, transmembrane transport is of great interest to us. The particular precursor of interest is salicylic acid (SA). SA enters the cell through facilitated diffusion as an uncharged molecule. It is deprotonated after encountering the cellular environment, affting different metabolic processes. We have been adding the sodium salt of salicylic acid, namely sodium salicylate. Thus, we can expect that a reduced number of salicylic acid molecules are entering the cell.

Experiment Description

  • We will change--particularly acidify--the pH of the media into which we add salicylic acid. We expect that, within tolerable conditions, SA uptake will increase with decreasing pH. We intend to measure SA uptake initially by noting the strength of the final smell compound methyl salicylate.
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