Lab 2

lab 2

Introduction

As engineers, synthetic biologists engage in the “design--> build--> test” process. They design genetic devices by coupling together promoters, ribosome binding sites (RBS), open reading frames (ORF), and terminator sequences. They then build these using techniques such as DNA synthesis, gel electrophoresis, polymerase chain reaction, and cloning. The synthetic biologists then test the function of the device using methods such as enzyme assays, fluorescent… and??? During the testing phase of this process, the synthetic biologists will characterize the device. They will measure and record the speed of response and level of protein production. One may be tempted to think that when designing genetic devices that a strong quick level of response is always desired. However, given the function of the device, it may be desirable to be able to tune the output to different levels. This may be accomplished through two means. One method is using promoters that differ in efficiency which will affect the rate of transcription. The other method involves using RBSs that differ in efficiency which will affect the rate of translation. Finer tuning can be achieved by the combination of promoter and RBS chosen. One device that has been constructed includes a stationary phase promoter, RBS, a lacZ ORF that produces beta-galactosidase , and terminator sequences. These devices all contain the same b-gal ORF and terminator sequence, so any difference in activity level will be due to the combination of promoter and RBS. This lacZ ORF provides us with an easy method to measure the activity level induced by each promoter/ORF combination. The beta-galactosidase that is produced by the lacZ enzyme allows the bacteria to metabolize lactose (see lac operon). However, if we provide ONPG (o-nitrophenyl-β-D-galactoside), it will be metabolized by the beta-gal into galactose and o-nitrophenol, a yellow compound. The intensity of the yellow color will be proportional to the amount of beta-gal that has been produced. In this lab we measure the changes in yellow color using a spec 20 as an assay of beta-gal production controlled by each combination of promoter and RBS.

Data Table

In your lab notebook, you will need to construct a data table as shown below. (These may be provided)

SAMPLE________________________________

Promoter	RBS	OD420	OD550	OD600	β-gal activity Miller Units	class mean β-gal activity Miller Units
weak	weak
weak	medium
weak	strong
medium	weak
medium	medium
medium	strong
strong	weak
strong	medium
strong	strong

Calculations

The β-gal production is reported in Miller Units

1 Miller Unit = [math]\displaystyle{ 1000 * \frac{(Abs_{420} - (1.75*Abs_{550}))}{(t * v * Abs_{600})} }[/math]

Where: Abs 420 is the Spec 20 absorbance at 420 nm. It is a measure of the yellow color produced by the β-gal activity.

Abs 550 is the Spec 20 absorbance at 550 nm. It is a measure of cell debris.

Abs 600 is the Spec 20 absorbance at 600 nm. It is a measure of the cell density.

t is the reaction time in minutes.

v is the culture volume in mls.