BE.109:Systems engineering/Advanced bacterial photography: 2 color
Introduction
One approach to understanding what nature has made is to build it yourself. Synthetic chemists may try to synthesize a compound from starting materials to test of their understanding of its chemistry: success offers additional support for the existing model while failure reveals its limits. Similarly efforts to build life from the bottom up can both confirm and advance conclusions drawn from deconstruction experiments that take a cell apart by mutation or through biochemistry.
Biology is particularly well suited to model building since many natural responses appear digital. One example of genetically-encoded digital logic is the heat shock response in bacterial cells. E. coli grow happily at 37°C. Shift them to 42°C and a group of genes, collectively called the heat shock genes, are induced to keep the cell from overheating. The speed with which the induction occurs is remarkable. Full induction is measured in cells after only three minutes at the elevated temperature, allowing gene expression to be modeled as either “on” or “off.” The heat shock promoter can be considered one processing unit that converts environmental information (heat) into transcription units. The heat shock gene is a second processing unit, converting the transcription units generated from the first “black box” into a protein.
The digital responses of cells to perturbations, combined with lab techniques for moving DNA parts around, allow logic functions and circuits to be constructed in living cells. Consider what would happen if the heat shock gene mentioned above was replaced with a gene for a repressor protein and, through a second manipulation, the repressor-controlled promoter was placed upstream of the heat shock gene. When cells bearing these constructs get shifted to 42°C, the repressor protein would be expressed and would turn off transcription of the heat shock gene. This precisely inverts the logic of the natural response when heat shock genes turn on in response to extra heat.
DNA can be programmed to implement other logic functions (AND gates, etc), enabling complex circuits to be built inside a cell. In principle, devices like the genetic inverter could be cleverly combined with other Boolean logic functions to design and synthesize a cell with predictable behavior. In practice, spatial and temporal factors hamper even simple designs. The cell is a messy circuit board without the static physical separation you could find between electronic circuit elements. Proteins are made and roam the cell, invariably interacting with nucleic acids and with other proteins in unpredictable and unspecified ways. A repressor protein might bind a non-consensus DNA elements for example, stressing or changing the behavior of the cell in some unanticipated way.
Circuit composition is also difficult because there is no standard expression level for genetic elements inside a cell. Genes are transcribed and translated at very different rates with wide variations in mRNA and protein stability. So while electrical engineers can find voltage gates that will work over a pre-specified range (0-5 volts for example) there are no specifications like these for biological systems. “Polymerases Per Second” (PoPS) is the unit that’s been proposed for measuring gene expression rates (recall your reading in comic ref) but operationally there is currently no simple way to measure PoPS. This creates a problem of “signal matching” as circuits are composed, since the output of one genetic device may be below the threshold of the one it’s designed to drive. At least for now, building complex circuits in cells is still a research effort, relying on trial and error to compose devices successfully. From an engineering perspective, it’s inefficient to try many combinations of genetic parts in realizing a design, akin to building a bridge in order to know if it will fall down.
In lab today you will invert the logic of the bacterial photography system, then begin your characterization of the system’s output, varying some experimental parameter to assess how that affect a part’s performance.
Protocol
Part 1: Advanced bacterial photography
The strain you will be using today is similar to the one used for black and white photography but in addition to the output generating device you are familiar with, a second device has been encoded: BBa_M30010. Look at this part at the Registry and try to predict what would happen under light and dark conditions.
| Condition | LacZ (on/off?) | cI (on/off?) | mRFP (on/off?) |
|---|---|---|---|
| Light | |||
| Dark |
BBa_M30000
BBa_M30010
The mRFP protein from basic part BBa_E1010 is detectable in ambient light as red-colored cells. You should use the dual reporter strain (red and black) to take another “biofilm.” You may use the same image as last time or create another one, depending on how happy you were with the results in black and white.
Part 2: Variations in operating conditions
The remainder of this experimental module will be dedicated to measuring the bacterial photography system output under experimental conditions of your choosing. Ultimately, you will document your findings at the Registry of Standard Biological Parts, allowing others to be informed by your experience with this system.
Think about what might be an interesting variation on the standard protocol and then plan a way to vary it. Experimental changes that affect growth might be good ones to try since cells will express unessential genes less well when they are stressed (by starvation, suboptimal temperatures, unusual pH etc). Other interesting experimental variations might relate to the light detection set-up itself, changing the intensity or spectrum of the light. Not every variation is possible but the teaching faculty will do their best to help you refine and then realize your plan. Before you leave lab today you should be growing 5 ml of the bacterial photography strain under two conditions. Next time you will compare the LacZ production (protein and RNA) in these cultures.
Part 3: Science and society
The remainder of the lab period has been set aside for you to think further about Synthetic Biology as an emerging technology and to discuss your ideas with the group. Begin by reading The engineer's approach to biology by Holger Breithaupt and Foundations of engineering biology by Drew Endy, and then consider what incomplete knowledge of biology means for efforts to engineer biology. How do engineers traditionally deal with complexity? Would similar methods work in biological engineering? What about the pressure that evolution exerts on living systems, particularly as they are strained and challenged? Do current risk estimates for naturally occurring biological organisms apply as well to engineered life? By the end of today’s lab you should have
- contributed to the conversation
- clarified your thinking about the challenges and risks associated with synthetic biology
- gathered some techniques for writing a persuasive argument
DONE!
For next time
Continue your writing assignment, focusing on your look to the future. Describe some of the best and/or worst applications of synthetic biology that may enrich/endanger society. Specifically you should make a chart. On the right side you should list 5 suitable, interesting, useful applications for synthetic biology. What would you love to see genetically programmable cells doing? Be clear about the benefit(s) these cells could offer. You do not need to figure out how you would make the cells do the things you propose (unless you want to). On the left of your chart, you should list at least one risks associated with each of the applications you describe. For example light-responsive cells capable of taking a picture may morph into light-responsive toxin-producers that could decimate Gotham every sunny day. Finally, make a wish list of at least 10 things that will be needed to achieve the applications you envision. These things can include improvements to currently available technologies, additional financial resources, improved understanding of biology, new trade/criminal/patent laws, changes to the social structure around research. Consider anything but be specific with your wish list (e.g. say “de novo DNA synthesis costs must fall below $0.001/base” rather than “more DNA synthesis”)
Next you should specify a part integral to your own system idea. You should retrieve sequence for at least one of the new parts you will include in your system and add the part to the registry, including "biobrick" ends and useful annotation as you do so. Print out the page with your new part number to hand in next time. If there are no new parts to your proposed system, or if the sequences you need to build with are not currently known, then use NCBI as a resource and specify something that is not currently in the registry but that might be useful to future genetic programmers.
