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H . T . G . A . A .   |   RESEARCH ASSIGNMENT   |   WEEK 1

SYNTHETIC MINIMAL CELLS

In this exercise you will use some light pipetting skills, you will prove you can work in RNAse free environment, and you will do simple reaction setup calculations. You will also need to look up few simple biochemical values in the literature.You will perform experiments that will result in direct visualization of the translation processes – observing the protein product as it’s being formed. You will set up one of two cell-free protein synthesis reactions: synthesis of GFP or FIAsH protein.GFP is green fluorescent protein – you can go learn more on Wikipedia. Mature GFP is fluorescent on it’s own – the fluorophore is made of amino acids from the GFP protein itself.FIAsH is a very short protein, not fluorescent on its own. The fluorescence comes from the ligand, a small molecule that’s bound to the FIAsH peptide. The ligand’s full name is fluorescein arsenical helix binder bis-EDT adduct, FIAsH-EDT2.Set up few GFP reactions and few FLAsH reactions, depending on how many people there is in class and how would you like to split into groups.


We set up wo cell-free protein synthesis reactions: synthesis of eGFP or FIAsH protein.
The reaction calculation from the group is summarized below:



For the wavelength analysis procedure, we used 470 nm excitation and 515 nm emission (by Priya) for the result due to the fact that we were unable to get result from the standard wavelength used (The excitation of eGFP is 490 nm and emission is 510 nm, as seen in the graph below. The excitation of FlAsH is 500 nm and emission is 540 nm.) Below are the graph of the results.

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