20.109(S11): M2 Temporary Space
Introductions Days 7 and 8
Utility of SB in general.
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.
Utility of modeling.
Protocol Day 8
journal discussion questions
descriptions/scope of figures etc
figure out what will be general discussion vs. assigned figs
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.
- 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)?
- 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?
- 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.
- 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)?
- Blue group: Figure 3 (focus on a-b, not c-d); 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.
- Pink group: Figure 4c and Emergence main text
- What effect does cell density have on system dynamics and why?
- 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?
- do two spins...
- Resuspend the cell pellet in 250 μL buffer P1.
- Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
- Add 250 μL of buffer P2 and mix by inversion until the suspension is a homogeneous blue colour.
- About 4-6 inversions of the tube should suffice. The blue colour comes from a special reagent that is not required for purification, but is simply used to check one's mixing technique.
- Buffer P2 contains sodium hydroxide for lysing.
- Add 350 μL buffer N3, and mix immediately by inversion until there is no blue colour (4-10 times).
- Buffer N3 contains acetic acid, which will cause the chromosomal DNA to messily precipitate; the faster you invert, the more homogeneous the precipitation will be.
- Buffer N3 also contains a chaotropic salt in preparation for the silica column purification.
- Centrifuge for 10 minutes at maximum speed. Note that you will be saving the supernatant after this step.
- Meanwhile, prepare 3 labeled QIAprep columns, one for each candidate clone.
- Transfer the entire supernatant to the column and centrifuge for 1 min.
- Wash with 0.5 mL PB, then 0.75 mL PE, with all spin steps 1 min long.
- After removing the PE, spin the mostly dry column for 1 more min.
- It is important to remove all traces of ethanol, as they may interfere with subsequent work with the DNA.
- Add 30 μL to the top center of the column, wait 1 min, then spin 1 min to collect your DNA.
Day 5 Intro
Last time you were here, you transformed your new construct — pED-IPTG-YFD — into XL1-Blue cells. As you can see in the linked manual (PDF), these cells have the following genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F ́ proAB lacIqZδM15 Tn10 (Tetr)]. Two gene mutations make XL1-Blue very useful "workhorse" cells for cloning. First, endA1 limits the non-specific destruction of plasmid (and chromosomal) DNA by the EndA enzyme, thus maximizing its recovery. Second, recA1 makes the cells incapable of homologous recombination, which could otherwise cause undesirable intermingling between the plasmid and chromosomal DNA.
You will extract DNA from three independent colonies that we picked and grew in liquid culture overnight. The procedure you will do is commonly termed "mini-prep," which distinguishes it from a “maxi-” or “large scale-prep” that involves a larger volume of cells and additional steps of purification. The overall goal of each prep is the same--to separate the plasmid DNA from the chromosomal DNA and cellular debris, allowing the plasmid DNA to be studied further. In the traditional mini-prep protocol, the media is removed from the cells by centrifugation. The cells are resuspended in a solution that contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. A solution of sodium hydroxide and SDS is then added. The base denatures the cell’s DNA, both chromosomal and plasmid, while the detergent dissolves the cellular proteins and lipids. The pH of the solution is returned to neutral by adding a mixture of acetic acid and potassium acetate. At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. In addition, the DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The plasmid DNA renatures normally and stays in solution, effectively separating plasmid DNA from the chromosomal DNA and the proteins and lipids of the cell.
Normally in 20.109 we do an in-house mini-prep procedure according to the steps above and followed by an ethanol precipitation step. However, because we are working with a low-copy plasmid, we will use a commercially available kit today to give you the best chance of success. The principle is the same as that of our "quick and dirty" (and cheaper!) prep, but is combined with the silica gel column purification you are familiar with from using other Qiagen kits.
We discussed last time that not all of your colonies may be carrying the correct plasmid, which is why you will isolated three separate clones. The most likely incorrect plasmid is the original pED-IPTG-INS, one that was singly cut (or less likely not cut at all) during the enzymatic digestion. Part of your miniprepped DNA will be sent for sequencing, and you will analyze the results next time to determine a clone to move forward with. In the meantime, you will transform all three candidates clones into the edge detector cell strain, JW3367c. In order to make our IPTG-sensitive results as comparable as possible to the light-based system, we are using this strain even though strictly speaking we don't have to. The genotype of a precursor to JW3367c can be seen at this link. Of particular importance to the edge detection system are the deletions of native lacZ and envZ, which could otherwise cause background production of black pigment or cross-talk in the 2-component signaling system, respectively. (Recall that the edge detector strain carries a plasmid with Cph8, a fusion of EnvZ and Cph1 that makes the former sensitive to light instead of salt concentration.) Before transforming JW3367c, you will have to make these cells competent to take up foreign DNA by treatment with calcium chloride.
In another week, you will finally get to see the results of all your hard work, and know how your modification affected the model system!
"The strain for all experiments in this study is E.coli JW3367 (E.coli K12 W3110, envZ-lacZ- NCBI-GI: 89110606) from which the Kanamycin resistance marker is removed (termed JW3367c)."