Final Project Homework

Main performance element to take into account:

Possible different elements, such as genetic design, regulation strength, growth efficiency, but it can all be constrained within one element downstream from all of them.

• Pigment production.

This can be determined as the amount (in metric weight units)of the coloured pigment produced by a specific culture of E. coli transformed with our plasmids of interest. Ideally this amount grows exponentially as the size of the culture grows, until large volumes require proper aeration and turbidity measurements to ensure the maximum efficiency of the process.

The best way to ideally determine the amount of pigment in samples would be by initially extracting the pigment from the producing cells by centrifugation of the culture, cell lysis and separation of the soluble pigment from cell debris.

In order to do something like that, an high-speed centrifuge would be needed for the initial spins of the cultures, proper lysis procedures, such as using 100% ethanol, which pigments are soluble in, and an eventual final centrifugation at highest or ultra speed to properly separate cell debris. The purified samples should then be analysed via LC-MS in specific amounts against a known standard to quantify the amount present in every sample, allowing for the determination of the ideal candidate strain within a library of possible producers.

Waters Homework

Part 1: Molecular Weight

• Based on the presented sequence for eGFP:

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VSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH

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The average molecular weight of the molecule would be 27875.41 Da, or 27875.41 g/mol.

• Calculating the molecular weight by using the adjacent charge state, if we use the provided spectrum:

Using the highest intensity peak n (824.1148) and its next charged state n+1 (849.0598), the mw of the protein is calculated as 27985.6560 Da.

However, in order to check the accuracy, other combinations of peaks should be tested and compared.

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This table shows that the calculation is fairly accurate and consistent within different peaks, and fairly close to the result given by the previous method.

Thus, the accuracy of the measurement would be of essentially 100%, as the value produced by the equation is 0.000004764.

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Part 2: Peptide work

Based on the above sequence, eGFP has 247 amino acids in its structure. Six of those are part of the His-tag used for purification, and since we do not know the size of the linker, we cannot exactly know where it starts and it ends, so with this information we can assume that eGFP has around 240 amino acids in total.