2020(S11) Lecture:week 2

Week 2 Tuesday

E. chromi

Let's start with an analogy that's a perennial favorite for engineers: consider a car....A car is a highly engineered system of interconnected parts. Many car parts are similar from car to car, but often the parts must be tailored to the size and function of the car. The chassis of a truck, a GTO muscle car and a Toyota hybrid are different, and so are many of the internal parts that make up the engine and the drive train. We might be able to move a radio from a truck chassis to a sports car chassis, but not much else. The car manufacturers are comfortable with this complexity, and it has little effect on the user of the car.

Now think about building a DNA program that runs a cell. We saw last week how a yeast gene could be moved to bacteria, allowing those cells to smell like bananas under certain growth conditions. From this one experiment you might be inclined to think that DNA parts will run reliably, independent their cellular context. Today you will explicitly test that notion.

Genetic Programs

You will compare the behavior of two genetic programs:

• pPRL, a purple color generator
• pGRN, a green color generator

These genetic programs were designed, constructed, and tested by the 2009 University of Cambridge iGEM team.

Cellular Chassis

In small groups, you will put these programs into two kinds of E. coli

• Strain 4-1, a K-12 strain of E. coli
• Strain 4-2, a B-type strain.

If you are curious about the ancestry of these two "breeds," there is an interesting article linked here. The short version of the story is that the K-12 strain was isolated from a stool sample of a dipthertia patient in Palo Alto, CA and domesticated for the lab by Tatum and Lederberg around 1922. By contrast, a B-type strain was used in the early 1900s by the Phage Group at the Institute Pasteur. Both types of E. coli are still widely used in research labs today.

Procedure

1. Begin by reviewing BioPrimer 7. If you'd prefer you can download it here.
   
   Is it clear what the differences are between the 2 strains of bacteria we'll be studying? What about the genetic programs?
2. Next we'll watch a short animation about the technique of DNA transformation. Is it clear what the steps are and why they are performed?
3. Finally, you'll work in small groups to transform the purple or the green color generators into Strains 4-1 or 4-2 as described here.
4. When you are done performing these manipulations, please wash your hands.
5. The petri dishes will be incubated at 37° overnight and you will examine the results of your work tomorrow.
6. Before you leave today, we'll consider these questions:

• Did you make any mistakes that might affect the outcome of this experiment?
• How confident are you in the results you'll see tomorrow?
  • Are you expecting colonies on all the plates? Are you expecting the same numbers on all plates?
  • Are you expecting the colonies to all look the same?
• If there are differences tomorrow, how will you explain them?
• If there are differences tomorrow, what could you do to test your explanations?

Why are we doing this?? You've taken some seemingly simple steps today and done something pretty awesome, namely intentionally imbued a bacterial host with properties you've chosen. Tomorrow, if all has gone well, you'll see colorful, antibiotic bacteria growing on the petri dishes. This technology has been used routinely for a generation or so. Consider, though, what it will mean, as we get better at both reading DNA programs that exist in nature, and also MAKING DNA programs that we dream up. DNA synthesis is a key enabling technology in synthetic biology, one we'll hear a lot more about tomorrow. In advance of that discussion, you might watch the DNA synthesis animation on the BioBuilder website, and also look at the journal article we'll be discussing. The article describes what the authors terms a "synthetic cell."

Why are we doing this?

Week 2 Studio

1. Next, upload your data to the BioBuilder website

Homework

Week 2 Thursday

Why are we doing this?