SEED/2012/Day 8

Design Project Group Assignments

Email Checkin (due Thurs 11pm)

Each design project group is required to check-in with the instructors via email, by Thursday night. We want to hear from you about the general state of your project, so we can make sure you're on the right track. You should mention your progress on the Device Diagram assignment below, as well as general project thoughts/updates.

Please CC all group members on this email.

Device Diagram

For this week, you will be drawing a device diagram of your system, which we will help you refine in class. You may wish to bring two copies of your diagram, one to turn in and one to make notes/changes on for later reference.

Start by considering the various functions your system will need, and separating them out into clearly defined devices. Next, draw (or describe) the interactions between those devices -- which ones turn other ones on/off and when?

Example devices:

• pH sensing device
• color producing device
• kill switch (device responsible for making the cells die when we tell them to, so they don't invade the patient or the environment)
• device allowing cells to survive in freeze-dried form, for convenient packaging
• timing device (see below...)

You should not have a device called "internal logic". The internal logic of your system is the combination of interactions between devices. You might have devices that contribute to the internal logic (i.e. devices that are not inputs or outputs), but they should still have a specific function. Likewise, if you have a "timing" device, ask yourself what the system behavior should look like over time and what inputs/conditions might count as start/stop signals.

Examples of internal logic devices: (we will go over some of these in class)

• "pulse generator", to turn another device on and then turn it off after a set time period
• "toggle switch", to switch a system from one behavior to another based on two inputs
• "oscillator", to generate a sine-wave function that will make other devices turn on and off at regular intervals

Individual Assignment

Building an Operon

THIS ASSIGNMENT IS TO BE TURNED IN VIA EMAIL, NOT IN PERSON. IT IS STILL DUE BY 10AM THIS SATURDAY. You should email your document to <seed-synthbio@mit.edu>.

In this assignment, you will be building an operon from DNA parts in the Registry of Standard Biological Parts, as we did in lecture last session. After you build your operon, you will answer some questions about it.

0. Open a document in Word or your favorite text editor that can do color highlighting. Use a fixed-width font such as Courier, not a regular font like Times or Arial.

1. Go to the Registry of Standard Biological Parts. Navigate to the "Catalog of parts and devices", which will be your starting point for the rest of this assignment.

2. The first part of an operon is the promoter. From among the promoters in the catalog, find the version of the pBad promoter that is NOT fused to the transcription factor araC.

Hint: the part should be 130 bp long.

3. Get the sequence of the pBad promoter. (When you go to the page for a specific part, you can get its DNA sequence by clicking on "Part Design" in the blue toolbar at the top, and then clicking "Get Selected Sequence" in the other blue toolbar above the cartoon drawing of the part.) Copy this sequence into your text document and color these basepairs green.

4. Next, find a strong Ribosome Binding Site (RBS) from the Community Collection: B0034, which has been chosen to define the "unit" of RBS strength. Copy/paste its sequence into your text file, placing it after the promoter. Color these basepairs light blue.

5. Now find the gene you want. We will use the araC gene, which codes for the araC transcription factor, which binds and activates the pBad promoter when arabinose is present. (Thus, our operon will become a simple system with positive feedback -- once it is turned on by adding arabinose, it will produce more araC that keeps itself active.) From the "Protein Coding Sequences" section of the catalog, find the version of the araC gene with an LVA tag. Copy this sequence into your text file and paste after the RBS. Highlight the start codon and stop codons yellow. (Do not highlight the rest of the protein coding sequence. Note that the LVA tag comes before the stop codons.)

Hint: You will have to find the right sub-category of "Protein Coding Sequences" that contains araC, which is a transcription factor.

6. To end the operon, we need a transcriptional terminator. Find the T1 terminator from E. coli rrnB. Copy this sequence into your document and highlight it red.

Questions:

1. Which part's sequence would you change to alter the translation rate of this gene?

2. If you made a lot of random changes to the pBad promoter sequence, would you expect transcription of this operon to increase or decrease? Why?

3. If you made a lot of random changes to araC gene sequence, would you expect transcription of this operon to increase or decrease? Why?