Measuring System Performance

Introduction

Operation of System's Devices

The bacterial photography system we are studying modifies a natural signaling pathway in E. coli so that light striking the cell can be converted to a detectable output (β-galactosidase enzyme that works on a chromogenic substance).

Bacterial Signaling for Photography System

2CS K+ and P+ activities

Black box abstraction

A “black box” depiction of the system looks like:

Some of the details within each box are:

This figure is more detailed but harder to decipher without prior understanding of several aspects of the system. For example, light at 660nm is detected by the input-sensing device, turning it off. Light-sensing requires the combined action of two sets of proteins: Cph8, itself a fusion of a light-sensing protein called Cph1 that comes from an algae attached to a transmembrane signaling protein called EnvZ, and phycobilin producing proteins that generate accessory pigments needed for the light-sensing protein to work. The input sensing device generates a signal within the cell’s osmoregulation pathway (OmpR is the signal carrier), changing the activity of an OmpR regulated promoter that is directing transcription of the output: lacZ….whew! The black boxes were a whole lot easier.

Today you will assess the system, both in terms of enzymatic activity measurements as well as a resulting bacterial photograph. Next time we'll start to "tune" the system in order to expand the dynamic range of the system.

Protocols

Part 1: β-galactosidase assay

You should perform assays on your overnight liquid cultures that were grown in the dark and the light. Refer back to the protocol from day 1. Activity calculations for today's samples will be part of your assignment for next time.

Part 2: Black and white photography

Retrieve the Petri dishes you set up last time and compare the appearance of the light and dark grown samples. Because the dark grown cells were in a completely dark box, the difference between the two plates reveals the greatest contrast you can expect in your bacterial photographs. You should take a photograph (digital!) of these petri dishes since the result will be included in the final research article you write for this module.

Next decide what image you would like to develop as a bacterial photograph. Generate a computer file with this image and print it to a transparency. Transparencies will be available in the lab for you to use as masks. As you choose an image, remember that the goal is to have each cell growing distinctly in the light or dark. Remember that light can bounce around edges and may blur the resulting image if the black and white are highly intermingled. In general, it’s better to have a dark background and a light image rather than the other way around. To darken the dark parts of your photo, you might want to print it on two transparencies and use them both to mask your Petri dish.

Media containing S-gal is available for you to supplement with antibiotics and cells as you did last time. Once you've supplemented and poured a plate, cover the dish with Saran (tape to keep in place) and use the point of some scissors to cut several slits in the saran. Next you can tape the transparency to the saran covering the Petri dish. Place your bacterial photograph in the 37° incubator (no lid) and when the class is ready, we'll turn on the red-lamp to expose the "coliroids" until next time. You will want to adjust the placement of the dish in the incubator once the light is on. The rule of thumb is the light image on the plate should be clearly visible but not super bright. If you can see the image clearly, and it doesn't look dim, you're probably in the right range.

Part 3: Instruction on Oral Presentations

Over the course of the term, everyone taking 20.109 will be making two presentations to the class. One will be a presentation of a primary journal article related to either 2 component signaling systems or synthetic biology. The other presentation will be of a research idea. Today our wonderful coaches from the Writing program will come to lab to give a talk about giving talks. This presentation should be a welcomed chance to learn what's expected when you speak (in 20.109 and elsewhere) and to gather some great tips for giving polished and interesting seminars. You can follow up this instruction by re-reading the oral presentation guidelines we've assembled for this subject.

DONE!

For Next Time

Calculate the units associated with the bacterial photography system grown in the light and the dark. Use this data and the photographs of your petri dishes grown in the light and the dark to draft Figure 1 of the research article you'll write for this module. Be sure this figure has all the traditional elements for figures (title, legend, labels etc).

Reagents