20.109(F10): Mod 2 Day 2 Measuring system performance

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20.109(F10): Laboratory Fundamentals of Biological Engineering

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Contents

Measuring System Performance

Introduction

In designing a synthetic biological system it can be helpful (to you and to others) to describe the system using a high level design language that can simplify or "abstract" what's going on with the system. For example the bacterial photography system might be depicted as two key devices, one device that detects a signal and a second device that generates an output upon receiving information from the first device. Such a “black box” depiction of the system's operation enables easy re-use of the devices by others as well as helpful specifications for the needed connections between the devices (versus the materials needed for operation within them). For example, light is the input for the bacterial photography system, but it's straightforward to retask the system as a chemical sensor instead: just swap out the first "input sensing device" from one that's sensitive to light to one that's sensitive to your chemical of interest. Similarly, the output of the bacterial photography system is a black colored product but imagine hooking up the light sensing input device to chemotaxis as the output...then the cells might swim to the light rather than turn a color in response.

"Black box" depiction of bacterial photography system
"Black box" depiction of bacterial photography system


Operation of System's Devices

In this module we will not be re-designing the system but rather tuning it to increase the dynamic range. Thus we'll have to look carefully inside the "black box" depictions of the devices and in this way become "device experts" rather than "system experts"--though a clear understanding of the system we're working with is obviously needed. To look in the black box that senses light, we'll have to learn more about the two component signaling cascades that the device is built from.

Two Component Signaling

As was mentioned last time, the bacterial photography system exploits a common signaling pathway in prokaryotic organisms, namely the "two component signaling" (2CS) pathway. As you learned, the two components are the sensor and the responder molecules and they are sensitive to changing environmental conditions. Hundreds of signaling pairs have been identified, and though variations are plenty, there is a common theme to the signaling mechanism the two components use. In general the communication of the 2CS pair is via a three reaction "phosphorelay."

Rxn 1: ATP + SensorHis --> ADP + SensorHis~P

Stimulation of the sensor by a ligand (or a change in the environment or a detectable cellular input) leads to a change of state for the sensor, e.g. it may dimerize, change conformation, etc. This altered state stimulates the autophosphorylation activity of the sensor and the gamma-phosphate from ATP is transferred to a histidine on the cytoplasmic face of the sensor. Thus another name for the sensor molecule is "histidine protein kinase" and when a number of sensor molecules are compared, the histidine that is phosphorylated can be identified based on conserved sequence motifs that surround it.

Rxn 2: SensorHis~P + ResponderAsp --> SensorHis + ResponderAsp~P

To transduce the signal from the sensor that's at the membrane to an effector molecule within the cell, the phosphate on the conserved histidine of the sensor is transferred to an aspartate molecule on the responder. The phosphorylation state of the responder regulates its activity. In many cases, the phosphorylated aspartate enables the responder to bind the DNA and regulate relevant genes, provoking a sensible cellular response to the initial stimulus, e.g. swimming to the good food, transcribing pores to let in ions, etc.

Rxn 3: ResponderAsp~P + Sensor + H2O --> ResponderAsp + Sensor + Pi

To reset the signaling system, the phosphorylation state of the regulator must be returned to its original set point. A phosphatase activity is associated with many of the sensor molecules in 2CS paths, enabling a single molecule to both enhance and diminish the signaling, consequently provoking and dampening the necessary response.


Inside the black box abstractions

The bacterial photography system we are studying modifies a natural osmolarity signaling pathway in E. coli so that light striking the cell can be converted to a detectable output. The green and orange dumbell-shaped cartoon in the figure (below) represents the input sensor. It's represented as a dumbell since light-sensing requires the combined action of two proteins: a natural light-sensing protein called Cph1 that comes from an algae (shown as the green lobe) and the transmembrane two component signaling protein called EnvZ (shown as the orange lobe). To fully function, the cell must also express phycobilin producing proteins to make accessory pigments needed for the light-sensing fusion protein to work. These phycobillins are shown in the box at the left of the signaling pathway and marked as "PCB" on the green lobe of the fusion protein cartoon. The Cph1:EnvZ fusion, called Cph8 by the authors of the paper, starts a phosphorelay within the cell’s osmoregulation pathway. OmpR is the response regulator and signal carrier, changing the activity of an OmpR regulated promoter that is directing transcription of LacZ. Finally the β-galactosidase enzyme from LacZ works on a chromogenic substance, Sgal, turning the media dark when LacZ transcription is high (i.e. cells are grown in the dark) or precipitating less color in the media when LacZ transcription is low (i.e. cells are grown in the light)

Returning to the generic "black box" description of the system, we can fill in a few of the details, in a way that emphasizes the device details and aspects that might be manipulated to tune the existing system. You can decide for yourself if this is a helpful depiction of the system. Remember that you would like to have an image that emphasizes means of tuning the system. In our experiment, we'll be focusing on the OmpR signal carrier between the two devices. There are other ways to affect the input and output responses though and you should consider how helpful (or not) this abstraction is. If you like the depiction of the system shown here, know why. If you don't, come up with a better one!

Details inside of bacterial photography system
Details inside of bacterial photography system


Today, we will assess the bacterial photography system as it was originally made. We'll measure enzymatic activity as well as view the 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, let it solidify ~30 minutes at room temperature then tape the transparency to the back of the dish. Place your bacterial photograph in the 37° incubator (media side up) 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: CAD Tool for Synthetic Biology

You will start a 2 day activity today that will enable you to visually construct (today) and then simulate/analyze (next time) the biological network that underlies the bacterial photography system. The tool we will use is called Tinkercell and is described in a Journal of Biological Engineering article that you can find here.

Get started!

Open the TinkerCell application and begin by familiarizing yourself with the basic operation of the program. In particular, try to use

  • the Molecules" and "Reaction tabs: try to select 2 proteins, e.g. a reporter protein or a receptor, and place them on the network canvas. Then connect the proteins with a 1 to 1 reaction arrow from the "reaction" menu. To change the identity of the object you've got on the network palette, use the cursor arrow from the toolbar. You can find this function as the 6th icon from the left on the uppermost toolbar.
  • the Parts tab: try stamping out a gene expression cassette, i.e. a promoter, an RBS, a protein coding sequence, and if you like: a terminator. The cursor will need to be reset between each addition to the network using the cursor arrow from the toolbar. There is also a useful "undo" function next to the cursor arrow, if you need it.

Instructions to visually build the bacterial photography system

Now that you have some of the basic mechanics in hand, try to visually construct the bacterial photography system according to the tutorial linked here.

  • You should start this project on a new canvas, that you can select from the File Menu as a "new graphics scene."
    starting a new network canvas
    starting a new network canvas
  • 1. Assemble reporter gene:Under “Parts” tab, place “inducible promoter,” RBS, and Coding icons on canvas. It may be helpful to select the cursor arrow (6th icon on the top bar, shown in red) to change parts.
  • 2. Align and name report gene elements:Drag items on the canvas next to one another to align them (they’ll turn red when they are connected). Alternatively, select the three parts and then use the “align” option from the drop down menu. Finally, click on the name and rename the parts to reflect what they are.
  • 3. Add transcription factors:From the “molecules” tab at the top of the page, select transcription factor, and print two onto the canvas. One will represent the phosphorylated form of OmpR, and the other will be the de-phosphorylated form.
  • 4. Visual appeal:From “edit” menu at the very top of the screen, choose “add decorator.” A pop-up screen (shown on the right here) will allow you to “replace” the OmpRp with a phosphorylated icon.
  • 5. OmpR kinasing and phosphatasing:From the “reaction” menu, choose “1 to 1” (green arrow above) and then click on OmpR and OmpRP to add a reaction for that conversion to the canvas. You can reshape the reaction arrow and move the reaction label by clicking on the cursor arrow (indicated by red arrow above) and then dragging the canvas icon.
  • 6. Regulate Transcription:From the “Regulation” menu, select the regulation icon and then click on OmpRp and the pOmpC box to add that regulatory reaction
  • 7. Add a Chassis:From the “compartments” tab, choose “cell” and print one on the canvas. Use the cursor arrow from the main menu bar to select the cell then move and resize it so it encases the transcription factors and reporter construct.
  • 8. Add the light sensor:From the “molecules” tab, choose “receptor” and print two to the cell’s membrane. Rename them Cph8 and Cph8p and add a phosphate decoration if desired.
  • 9. Add regulatory reactions:Use two of the “1 to 1” reactions to connect the receptors. If you are having trouble selecting the objects on the canvas, you can zoom in or out using the magnifier icons.
  • 10. Connecting to the response regulators:Using the “regulation” reaction, connect the phosphorylated form of Cph8 to the reaction arrow that converts OmpR to OmpRp.
  • 11. And let there be light:From the “molecules” tab, choose “small molecule” for the canvas. Rename it “light” and add a “1-1” regulatory reaction from the light triangle to the reaction that converts OmpRp to OmpR
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You can save your work at any point, but should ideally, complete the exercise today. No matter how far you get, be sure to save the file in order to run the simulation and analysis of the network that you'll try next time.

Whew!

Now that you've dragged, dropped and connected all the parts in the bacterial photography system, it's time to consider what you've got. Did you learn more about the system by building it? Can you see places where different parts might be interesting to try? How big is the gap between the things we know about, and the things we need to learn more about? Are there any experiments you'd like to try? What reactions could you measure? What could you mutate to improve?

Part 4: 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

  1. 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).
  2. Consider again the depictions of the photography system that we've looked at (system level depictions, biology cartoon, device level operation). In particular, think about how you might use the image below to describe the experiment you'll be doing. Is there a better depiction for the system and the aspects of it that you'll be manipulating? If not, say why. If so, sketch it out and say how it's better. There's no "right" or "wrong" here but it's important to think about effective figures that summarize and anticipate your work.

Reagents

See Mod 2 Day 1.
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