20.109(S11):Assay modified model system (Day7)

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20.109(S11): Laboratory Fundamentals of Biological Engineering

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Contents

Introduction

The concept of engineering biology, as opposed to investigating it, is still a relatively new one. While the boundary between the older field of genetic engineering and the newer one of synthetic biology can be fuzzy, the difference can perhaps be summed up intuitively in one word: more. Synthetic biology is more ambitious in scope, more systematic, and thus more reliant on tools and knowledge that don't yet exist. Projects range from fundamental proofs-of-concept to the highly application-driven: these include engineering yeast to produce an anti-malarial drug, finding the minimal gene set of an organism necessary to sustain life, and of course getting cells to carry out logical computations in genetic circuits. Many more examples can be found in synthetic biology reviews or in a reading list for a class taught by my colleague Natalie Kuldell. There is also a lot of information to be found on OpenWetWare, an initiative of the synthetic biology community in the first place!

As Professor Weiss discussed in class, the success of synthetic biology is dependent on gains in both foundational technologies (described here by former MIT Professor Drew Endy) and fundamental knowledge. In the former category, the cheap, reliable, and large-scale synthesis of DNA is a crucial one. Conceptual 'technologies' borrowed from other engineering disciplines, such as abstraction (away of details), are also important. Realistically, however, for now it is very difficult to design and make predictions about novel biological systems without understanding the component parts at a pretty sophisticated level of detail. Instead of taking an entirely rational engineering approach, scientists can also use directed evolution of existing systems in concert with (or independently of) rational design.

While the field of synthetic biology spurs on interesting and fun science, it also provokes worry. In a world (like deep movie voice "In a world... ") where biology is easier to engineer, will some bad actors take advantage of its newfound reliability and accessibility to make dangerous new lifeforms? In fact, DNA synthesis companies routinely screen for pathogenic sequences in their requests, a seemingly sufficient safeguard at this stage of the field's development. The ethical implications of synthetic biology are routinely discussed by the community as the field evolves, with the hope that standards can be developed largely from within.

Protocols

Additional part: tissue culture demonstration

Today we will be somewhat equipment-limited, as we only have one light box, one fume hood, and two spectrophotometers. For this reason, half of you will join me for a tissue culture introduction at the beginning of class, which would normally be done on the first day of the third module. This approach will introduce a natural staggering of equipment use. The second half of you will join me once you have all finished Parts 1 and 2, giving the folks who started later a chance to complete those parts. Then we will all come together for the journal article discussion, hopefully by ~ 3:30 pm.

Getting the (15-20 min) TC demo out of the way will also give you a chance to take in the techniques before you practice them (on Day 1) and implement them more carefully (on Day 2), as well as free up extra design time on Day 1 of the third module.

Part 1: β-gal assay of modified system

  • Follow the protocol from Day 2 for performing β-gal assays. When your reactions are stopped and spun down, transfer to a 96-well plate (previously described as Option 2). Files with your OD420 and OD550 values will be posted on today's Talk page.
    • Remember to order your samples from least to most expected activity, especially if one person is working without a partner during the ONPG addition step.
    • Order your samples in the first two columns of the plate in whatever order you like, but please put your blank is the first row of the third column.
Tube # Well # in plate Sample OD600 Time started Time stopped Time elapsed
(calculated)
OD420
(from file)
OD550
(from file)
Units (calculated)
0 3A 0:00
1
2
3
4
5
6
7
8
9
10
11
12
(13)
(14)
(15)
(16)

Part 2: Observe solid cultures of IPTG-sensitive systems

  1. Have a look at the four plates you prepared last time.
    • Does the IPTG spot result in the truth table value you expect for each plate?
    • Do the INS and YFD samples look different?
    • Within the same cell strain, do the plates with and without AHL look different?
  2. Take a picture of any plates that you might want to include in your report, or at minimum the plates with both IPTG and AHL spotted on them.

Part 3: Journal article discussion

Preface and Goals

The Hasty lab paper is probably more difficult for most of us to understand than was the paper we read in Module 1. For this reason, we will very much be approaching the figure assignments and resulting discussion as a collective attempt to elucidate the paper. Your brief presentation of a figure/topic from the paper should demonstrate that you have become familiar with it as best you can, but it's okay not to understand every detail. You may find that another group's presentation helps make sense of a piece you can't yet grasp. You should also feel welcome to switch figure assignments with another team, if you each have expertise (e.g., background in modeling or microfluidics) that may help you present each other's section. The important thing is that everyone is responsible for a careful reading of some part of the paper.

General Discussion Points

  • Introduction: How do the authors frame their topic and promote interest in it?
  • Results: Spatiotemporal data can be difficult to present clearly. What do you think about the data presentation in this paper? What would you do the same and/or differently?
  • Discussion: What is the usefulness (current or potential) of this work? What are some limitations?
  • Methods: What might you include for sections on plasmid construction/plasmids used/strains used in your own paper?

Assigned Parts to Focus On

You may all refer to the relevant supplementary movies for your sections as well.

  1. Red group: Figure 1a; later Figure 4a and Supplementary Modeling section
    • Describe the genetic circuit and how it functions.
    • At a high level, what features are incorporated in the model and how? What is the main new feature compared to previous work modeling oscillators (see main text)?
  2. Orange group: Figure 1b, Supplementary Microscopy and Microfluidics section, Supp. Figure 4a
    • Describe the physical set-up for the experiments and how it works.
    • What considerations affected the design specifications of the chamber?
  3. Yellow group: Figure 1c-d, Figure 2a-b, Supplementary Data Analysis section, Supp. Figure 2
    • What principles underlie the dynamic behaviour of the system in general (i.e., why are there oscillations at all)?
    • Discuss the data collection and processing.
  4. Green group: Figure 2c-d; later Figure 4b
    • What wave parameters vary with flow-rate?
    • Briefly, what causes 'degrade-and-fire' oscillations (reference 44 of the paper can be found here)?
  5. Blue group: Figure 3a-b; Supplementary Space-Time plots section, Supp. Figure 3
    • What set-up/parameters were different here than in Figure 1?
    • Discuss the main features of the resulting dynamics.
  6. Pink group: Figure 3c-d; Figure 4c and Emergence main text
    • Briefly discuss the 3D system dynamics.
    • What effect does cell density have on system dynamics and why?
  7. Purple group: Figure 4d, Supplementary Figures 5 and 6 and Emergence main text
    • Discuss the relationship between diffusion (of what?) and velocity (of what?) in Figure 4d.
    • What other effect(s) does changing the diffusion coefficient have under various conditions?
    • What is one thing that modeling allowed the researchers to learn that they couldn't access experimentally?

For next time

  • You are encouraged to calculate the Miller units for the assay you performed today, as you will be working with all of your data (as well as — how can I put this — making up some feasible data) next time. However, you are not required to turn anything in, and could potentially finish your Miller calculations at the beginning of the next lab session.
  • You are also strongly encouraged to check your Miller calculation process with the sample data below, so we are all on the same page for class-wide comparisons of data.
    • The culture volume used was 50 μL of a 1:5 instead of a 1:10 dilution.
    • The starting time is 90 seconds and the ending time is 2 minutes and 50 seconds.
    • The OD600 of a 1:10 dilution is 0.204 (read on the spectrophotometer, path length 1 cm).
    • The A420 and A550 values are 0.2616 and 0.0043 (read on the plate reader, path length 0.5 cm).
    • You should calculate about 18,700 Miller units for this example.

Reagent list

As on Day 2.
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