BioBuilder Workshop 2012

Day 3 Notes

Goal for the week: Find out whether and how BB can be taught at the MS level.

Introductions.
Today will be super busy :)

Morning: smell lab

Afternoon: color lab
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Jo-Anne and Anndy present Intro to Synthbio

Before I took the workshop, I thought I knew about biology and genetic engineering. Then I found out I had a lot to learn :)

Video clip: Youtube "Synthetic Biology Explained". (This is basically a standard explanation, but very well executed. It includes summaries of several iGEM projects -- E. chromi and bacterial photography. Also mentions Keasling's and Venter's work very clearly.)

I taught it to my regular students and my AP students, and even brought some of it to the middle school. They all thought it was amazing. I showed my students this video and their eyes lit up. "What about this? What if we could do that?" And things that won a Nobel 40 years ago, we can now do in a HS classroom.

Natalie's example of her plant dying when she goes on vacation. Proposing solutions from different engineering fields. Contrast traditional genetic engineering way vs. synthbio way of standardization, abstraction, synthesis.

I'm not an engineer or tinkerer myself, but I get it. Last year I was unsure, but I kept my mind open and now I'm totally sold.

Synthbio shows a connection between biology and engineering. Emphasis on "does the system work" instead of "I have to understand how every little piece works".
Anndy:

When I first learned about synthbio, I was thinking "cells don't do that!", but I found that saying "well, why not?" is very freeing.

Synthbio uses the principles of abstraction, modularity, standardization, design and modeling.

Abstraction: Use parts without knowing how they work. You can drive a car without knowing how it works.

DNA makes parts, parts make devices, devices make a system.

Example: A clock is made of parts -- motors, springs, plastic -- which could be recombined to make some other thing, like a toy boat.

Standardization: All the parts are interchangeable. For example, light bulbs and sockets. Incandescent or CFL or LED all fit the same socket. All the Tab A's fit in all the Slot B's.

Design and modeling:

Propose a problem

Think of a solution

Build a model

Test the model

Analyze the results (There's a chance to learn science here! If it doesn't work, could our basic biological assumptions be false?)

Improve design

Vocab terms:

The bacterium is the chassis

Parts are assembled from DNA. We can use parts from any organism (unity of life).

In cells, DNA is assembled in devices. For example, the lac operon! This is an existing device, made by evolution. The idea in synthbio is that we decide what each part is, and we make the device to make the cell do what we want.

Parts can be bought online -- Registry website.

DNA can be dehydrated and sent in the mail! Convenient!

Jo-Anne: I asked my students to come up with a device. They said "lead testing", and I clicked around on the Registry and it was all there, all the parts I needed. And I could get it shipped.
Anndy:

An example part is a Tet repressor promoter. We buy the promoter with this DNA sequence, an RBS with this sequence, an ORF which is the coding part of the gene, and transcritipnal termnators. We buy them and glue them together molecularly.

Abstraction hierarchy. [I missed some of the talk here]

And that's what we're going to be doing this week. It's awesome. Questions?

Jen: It's not that I don't buy it, it's the controversy. If my community garden friends knew I was here, they would hate me.

John: I get how it's fine not to know how it works and just put a part in, but for safety, at some level someone's gotta know what's going on. I know we're doing bioethics tomorrow...

Natalie: Yeah, we'll talk about safety tomorrow. I get on an airplane and don't know how it works, but I trust it. Biology has a special place in safety discussions because of the potential size of repercussions.

John: My MS kids see a lot of stuff in the media, and I spend half my time talking about how movies overblow stuff. But they are thinking of it. It does get them thinking.

Natalie: It's important to note that in a complex system there's no individual that understands the whole integrated system. People have individual expertise. So you need a good conversation. If that doesn't happen you get stuff like the Challenger disaster with the o-rings. So the idea of abstraction does have certain requirements.

John: The UMass library tower got vertically compressed, and bricks were flying everywhere, because the engineers forgot that books have mass. :) I see where you're going.

Lisa: It helps the creative process. We usually teach kids not to do something if you don't know what you're doing. But this goes against that tendency and facilitates creativity.

Marianne: My bioengineer friend spoke to my students and was talking about putting an engineered bacteria in the water that would detect mines. People were concerned so they tested and tested it and had them all die on the 6th generation. I thought that was interesting. In listening to your presentation, it's really thrilling to know that... you want to end on a high note, and there's a lot of gloom and doom in the newspapers with global warming, and it's very refreshing to think that I can say look, people are working on solutions. They have a real serious hope and you can be part of that. It's not just writing letters and stopping emissions. There's real possibility and flexibility.

Natalie: Yeah, there's hope that biology can meet these challenges, but we have to think carefully about safety.

Anne and Linda present Eau d' Coli.

Anne:

I thought about what I would do if I would show this to my MS students, so we broke it into a simplistic approach.

Black box, magic wand, come out with a product. Like the magicians we don't really know what the magic is, but we get the design out.

System level description: E. coli bacteria acts as a black box to do the work to perform the function of the system.

We need an input, and then out comes an output.

So the question was how can we alter the smell of bacteria. E. coli become our precursor compound and what we want to do through black box functions is convert that to a banana smell.

What goes in the black box?

We know from science that bacteria have three growth phases, lag log stationary. We put one bacteria in the black box and there are many coming out the end. [a very confusing graphic here...]

(explains stationary phase and population saturation)

Now we have E. coli and we put the banana generating device in the black box. Put it on our device list.

Two ways to do this: you can put isoamyl alcohol in the media or put in an isoamyl generating device. So now we have two devices in the box.

Additional part needed for activating banana smell in stationary phase. There's a promoter osmY that is active in stat phase. Turns this switch on only when there's a lot of bacteria. osmY added to BGD to make the switch turn on.

Three devices: isoamyl generating device, osmY, BGD. We start with nasty bacteria in lag phase and they grow and we get nice banana smelling cells in stationary phase.

The overall equation is: isoamyl or isoamyl-GD + osmY + BGD = isoamyl acetate. In the lab you won't have the IAGD. Isoamyl alcohol is in the media which will be converted through the BGD into isoamyl acetate.

Linda:

Who did this? Students. 2006 iGEM MIT undergrads. They were able to put it together. Maybe not a deep question but it was something they could actually do. Many hours of redesign, trial and error, using engineering design process.

John: So we peeked inside the black box... do you talk to HS students about sequencing? If the parts are assembled in the wrong order, it doesn't work.

Jim: With these HS teachers at the workshop, we wanted to do something that would allow you to approach bioogy through an engineering lens. You can't ignore the biology. As we teach it, we're able to review or teach a topic like operon structure. We're using it to do both, to look at basic molecular stuff and then talk about what if we swapped in a new one. We're not leaving it as a black box.

Sam: One of my inital concerns is, how can I make this inquiry based? I don't want to emphasize the "push this button and it just works" thing. We have to build in design so it isn't like a cookbook lab.

Natalie: Right after lunch we'll have people talking about using this approach and nature's parts to make something and then learning the biology content. I've found at MIT, when students think about which piece of DNA should go first in a construct, you have to know about these switches and genetic elements. That comes out of the desire to build something out of them. It gets to the scientific content. At the Berkeley workshop someone wrote that it is an atypical approach to ask students to do a lab and not understand 100% of everything going in. But it is like real research in that sense.

Linda: And that is like real engineering. You don't know all the aspects of the project going in, you don't know what will work best.

Sam: "Experiment early and often" mindset? As you go further down the line it's harder to change things you set earlier.

Natalie: Yeah

Jim: Using the "students design the experiment" definition of inquiry, it's hard to make it inquiry-based. You can't reengineer the system in the classroom but you can change the experiment a little. What's important is after the lab the students think of design idea what-ifs and discuss them. We'll talk about that tomorrow.

Sam: Does this work in a flipped classroom?

Natalie: We spent a couple hours yesterday talking about website resources that would be useful for a flipped classroom.
---

Sherry and Jo-Anne introduce the actual banana smell lab.

What was their purpose in developing the banana generator?

Marianne: To see if they could do it.

Sherry: E. coli smell awful. Here's an opportunity. We're going to learn about the generator and some competing designs.

Lab objectives:

Explain growth curve

Culture bacteria

Collect and analyze qualitative/quantitative data

Understand terms like part, device, inverter

Review growth curve: What happens during lag phase?

John: Slow growth.

Log phase?

Lisa: Dividing.

Stat?

You will have one sample of each phase for 4 strains.

Now to learn about the banana generating device.

Jo-Anne:

This symbol stands for a promoter. The promoter says get ready to read, initiates transcription and translation. Here is the RBS where protein synthesis starts. Here is the ORF, the gene that encodes the something, which is the protein, which is ATF1, which is the magic protein that cuts isoamyl alcohol in the media into isoamyl acetate, which smells yummy. Now I warn you, you will still get some of the icky smell. It's not gonna be a smoothie.

So this is what the whole system looks like. This is the original design.

Your challenge: We have these 4 strains. Strain 1-1 smells like bananas during stationary phase. [draws growth curve on board] Strain 1-2 is the original device with an inverter. One of the objectives was to understand what an inverter is. The inverter is like the switch, backward. The switch makes the isoamyl alchol be cut during lag and log but turns off in stationary.

John: So it's like a timer?

Jo-Anne:

Strain 1-3 has a log phase promoter. So instead of smelling during stat, it smells during log phase. Strin 1-4 has no smell generating at all. Why? Negative control.

Our challenge, the inquiry and engineering part, is to test each BGD to see which makes the strongest smell [I thought the goal was to test competing types of regulation? but the why is kind of unclear in this lab anyway.]. This is where we test our part.

Jen: When you put the inverter in, is it in the original device?

Jo-Anne: [something....]

Jen: So it's changing the function of the original switch.

Jo-Anne:

When I did this with students, I did all the culturing part. But for my AP class, I had two students do the culturing as an independent project when their own project didn't pan out. They did a good job. I told them along the way, now we're doing this, now we're doing that. This is how we get lag log and stat samples. Prepare cultures in liquid broth. Take some in the fridge before they hit log growth, and that's lag phase. Then we let it grow for 6h in the frige, log phase. Then let the others grow overnight, those are stationary. That's how you get the phases. We'll provide them all to you for you to smell. You get 12 samples.

For each sample you measure 2 things. You could use a spec or you can do it qualitatively. You can smell them and rank them. How will you measure smell? How will you standardize it? This is an engineering inquiry. You can let the students come up with the scheme. Does everyone smell it the same? You will also estimate cell population using McFarland standards which we will explain later. And we have banana standards, banana oil and water. It's qualitative. I found it helps to smell coffee in between banana samples, to cleanse your nose.

Recording data: Try this banana chart. Draw bars through a number of clip-art bananas. Then you flip it and it's a graph. A nice visual.

Jen: How to make the standards?

Jo-Anne: It's recipe on the website

Jim: Banana standards are good for months. Last year's have gone rancid. A year is no good but it's more than a week.

Jo-Anne: If you seal the tops it's fine

Jim: The turbidity standards last forever

Sherry: Open your binder to lab 1, see the links, this is a wiki page, takes you to protocol A or B. One has a spec and one doesn't. And also point out at the top, there are procedure videos. For teacher or student prep there are many video procedures. We'll talk about the setup after the safety training.

In lab.
EHS presentation.

Curriculum development discussion session. Teachers in groups, Jim sets forth goals: discuss and clarify understanding of material, think about how to use synthbio concepts in teaching.

After free discussion

[some stuff I missed here]

Jen: Ground it in the organelles, what 7th graders know about cells.

Natalie: The quantitative vs qualitative discussion comes up a lot. McFarland vs spectrophotometer. People are more confident in spec results. Andrea had an idea about fruit flies?

Andrea: I played with that, I'll have to see if I can dig up the data.

Natalie: Use it as a teaching point.

Natalie: That's a lab question. What about concepts?

Anne: We discussed correlating turbidity with the wow factor of how many cells are in the tube. These McFarland standards, can they be in the kit?

Natalie: That's hard for EHS reasons, with the barium. We might find a work around.

Lisa: Also the smell lab is like a big picture of engineering.

Natalie: People want to know which one comes sequentially after the smell lab, but they're modular. But in terms of getting what a synthetic system looks like, a banana bacteria is something you're not going to find in nature.

Mike: Well it depends if we are making a whole synthbio curriculum or choosing synthbio labs that can amplify or relate to something we're already doing.

Natalie: That's what we want to see from you on Friday when you report ideas. Having you here is our chance to learn what is possible. I don't know if it's possible to make a whole synthbio curriculum for MS or if it's better to view existing MS curriculum thru the synthbio lens. I'd be happy either way, even if the students aren't ready for it. You guys are just getting started. On friday I want to hear your ideas explicitly.

Lisa: Important to get students to understand the breadth of synthbio's applications vs. the one narrow spot I've put it in my class.

Natalie: You may think this isn't going to fit a great need of mine, but later you may think about refactoring a lot of things. It's for you to tell us where it fits.

Marianne: It takes a lot for students to understand that genes are not just hair color or the shape of your nose. You have to teach the basics, cells, organelles. I feel bad teaching them ribosomes though, because they're thinking, who cares. How many will remember ribosomes? This lab actually shows it in action, and that is elegant. It makes everything valuable.

Natalie: When I teach my lab class we design a PCR primer and for the first time everyone's like "oh, that's why we know about 5' and 3' ends". There's an actual reason you need to know.

Marianne: And when they come up with their design ideas it'll still be related to the ribosome!
Lianne: I teach physical science so it's challenging for me to integrate this, but I also have to teach engineering for MCAS. With this, I can show them it's not just about bridges and houses, it's also biology. Showing the banana smell is great.

John: It's also a really cool way to look at experimental design.

Natalie: [...] You can bring synthbio in whole hog or use it as a lens on your existing material... One thing you could do is just appreciate the currentness of these open questions. Not everything in science is finished.

John: I like to emphasize that everything I tell them is a simplification. You're not ready for it yet but you'll get there. Sometimes I show them advanced videos and point out the simple parts. Instead of just putting in the pGLO gene, this banana thing is a designed gene.

Jim: You'll love the next lab!

Natalie: So, be thinking about concrete things you could report on Friday. And how we can help. I don't want it to be so amorphous that I can't help. It would be great if you leav e here wth something you can try.

Sung and Andrea talk about chassis selection and synthetic cells

We took much of this from the BB website and Ginkgo Bioworks. Anything with the ginkgo leaf on it is attributed to them.

Quote: "We work on the edge of knowledge". This quote makes me feel comfortable not knowing everything and there are a lot of black boxes out there. That's good, we'll always be in business :)
Sung:

I think it's useful to show natural organisms with crazy remarkable behavior first, and then talk about how you can sequence them and put their genes into a standard chassis to manipulate them much easier.

Natural organisms: Cupriavidus metallidurans, there is a modified version of it that produces fluoresceins in presence of gold, so it's a mining extraction tool.

Deinococcus radiodurans, I spent half a year trying to sequence it, it's hard to get the DNA out. It has insanely robust DNA repair, you could throw it in a nuclear reactor. About 1000x as tolerant as a human cell.

Acidithiobacillus ferrooxidans. Used in bioleaching process where they separate out copper or other metals from impure ores. About 25% of commercial copper production involes bioleaching.

Spiderweb: Lots of interesting properties, strong and light and so on. But you don't want to farm spiders. Would be inefficient and slow. So people made the spider silk producing goat. The silk gene from spiders in goats and they produce it in their milk. You can process it to spider silk that's basically the real thing. It's faster and more scalable.

This is E. coli with ces7 protein, a heavy metal sequestering protein that turns the metals into quantum dots, which have many applications in imaging and so on.

This is the operons of firefly and jellyfish in E. coli, and as a joke they put the bacteria in lightbulbs.

Marianne: Do you have to supply nutrients?

Sung: Yes, it's LB media in there.

Andrea: These are just cute containers, not working light bulbs?

Sung: Yeah, this is a proof of concept.

Marianne: If they ran out of food, could you put a food making organism in there?

Lisa: Or a food substrate

Andrea: Or a photosynthetic thing!

Sung: That's the kind of discussion we're going for! What if you engineer a phototroph to glow? They'll survive and then glow at night.

Cambridge e-glo light 2011 iGEM project.
Andrea:

Now we can use living things to build, and engineer them the same way we engineer machines. We have certain methods for that.

Machines:

Have interchangeable parts. Can easily replace something if it breaks or if you want a different function. Modular.

Predictable. We hope they always behave the same way.

Are well characterized -- know the speed of a car, the heat of a toaster

Meet industry standards for safety and durability... planned obsolescence

Are safe

Difference between genetic enginering and synthbio... genetic engineering modifies one organism for one purpose, from scratch. Start with a species that's close to what you want and tweak it. For example, putting GFP in E. coli.

Whereas synthbio... Youtube video "Synthetic Kingdom". Talks about finding genes and putting them into plasmids and into chassis. Minimal genomes and minimal cells. And how this will form a new "synthetic organisms" cladistic kingdom... very futuristic and modern-art-y :)

Andrea:

Jen: Framework it sits on top of

Andrea:

This lab looks at choices of chassis. If you have different chassis will you get the same outcome? Intuitively, probably not... there are some surprising results. In synthbio, we start with a standardized chassis and add different parts and get different engineered organisms. If needed, we mgiht develop better standard chassis depending on what you're looking for.

Safe. Protect ourselves, environment, people we work with.

As simple as possible. If you start with something complicated, you get a lot of big and complicated things.

Genome sequenced and genes characterized

Engineerable

Intellectual property issues... e.g. Monsanto's terminated crops... checkpoints on these issues.

Don't want them to escape, make them weak in some environments. The Intel science fair now has a BSL1 safety checklist. You have to have screens on windows, that is new this year. That is to control insects coming in and walking on your plates and tracking them elsewhere. John Glass talked about how he's not concerned about engineered organisms being stolen, but people walk out with them on their clothes.

Well-behaved. No unexpected side effects.

Sung gave us great extremophile examples. Also look at Microbe Zoo, a fun website. You can have students do some bioprospecting and find chassis. Even relates to astrobiology. Go to Microbe Zoo, you can pull up these organisms, and think, what would this be a good chassis for? Or you can go parts mining in the Registry.

Craig Venter's research yacht. They are collecting stuff and sequencing everything, to find potential parts and chassis. Good interdisciplinary potential with social studies?

One consideration is simplicity. E. coli is standard but some folks want to start very simple. Mycoplasma and Mesoplasma have a few hundred KB genome, about as simple as it gets.

Broad institute is working on Mesoplasma florum.

Some reasons why M. florum is good: is BSL1, no growth at 37.

Why would no growth at 37 be a positive attribute for a chassis?

John: It won't grow in our bodies

Andrea: Right. Also, it grows quickly. JCVI's first organism took 3 weeks to double, which is undesirable. Think fruit flies instead of elephants.

Mike: Earlier your slide said "unusual food base" was a good criterion. Why?

Andrea: If you want to make an organism that eats something unusual like oil, you would have to pick a chassis that could handle that.

Lisa: Does that also have to do with control of escape?

Andrea: Yeah, nutritional mutations are a mechanism for control.

Sung: For example halobacteria, only survives in salty conditions.
Sung:

M. florum is well charaterized. Any chassis has to be sequenced, otherwise it's unpredictable and doesn't fit the engineering ethos.

You also want: easy to modify and transform, which is why E. coli is popular, it's easy to grow and transform.
Andrea:

Have you heard of Synthia?

Marianne: I read about it...

Andrea:

Marianne: It's still a naturally occuring cell, just scooped out.

Andrea: Yes, machine made DNA stuffed in there.

Marianne: When I tell my students we have never created a cell from scratch, that is still true?

Andrea: Yes. I think that's going to be a long time coming. But this was kind of a proof of concept. They had t find out if there was something special about the original DNA that we didn't know. Is a genome an interchangeable part? How modular is modular?
Video: Craig Venter's TED talk on synthetic life. Announcement of M. capricolum genome transplant success.
Andrea:

BB uses E. coli. But there are many strains of E. coli, like 70-odd commonly used.

They differ in pathogenicity and other ways.

Your turn: What chassis would you pick for these applications?

To synthesize human insulin: What was the first one they used? E coli. I think.

Sung: They did. Genentech. It was their first product. Large vats of bacteria in a bioreactor to pump out human insulin.

Andrea: Some of our vitamins are made by bacteria or yeast. A lot of biotech companies are using insect cells.

The bad idea game. What devices would you advise NOT to put into these chassis?

E. coli...? [no response]

Or corn?

Andrea: Yeah, the butterflies that eat corn pests and so on are being impacted. Lack of orthogonal design.

Questions?
Jen: So what would you not put in a chassis?

Lisa: Well you don't want them to be able to tolerate temperatures beyond the norm so they don't move into such environments. Or insects resistant to insecticide.

Sung: Another example would be UV resistance from radiodurans. A lot of industrial-scale disinfection is done by UV. If that goes into E. coli and to a pathogen then we're in trouble.

Marianne: Some of the vocab in this presentation, I would want to be careful with it with my students. Venter said this is computer code, and four bottles of AGTC. Is the computer putting drops into a tube...?

Andrea: That's kind of how a synthesizer works, with four bottles. You can synthesize about 500bp and then you have to connect them. So the technician would type in, make this sequence, and the machine puts it together.

Marianne: Using the chemicals in the bottles?

Andrea: Yes.

John: What levels of organism are we doing this to? E. coli, corn... up to what level?

Sung: Every single model organism in synthbio is a natually occurring organism, fully sequenced.

Andrea: So probably tons of variations of food crops. I mean, like, chipotle advertises that they do not use GMO corn. It's in the food chain.

Sung: In terms of easy chassis, Arabidopsis. High schoolers are doing stuff with Arabidopsis.

Andrea: My students have done it

Marianne: The chassis is one thing, but the stuff you put in it...

Andrea: Well you find it in the registry. By the way, I found some biobuilder candy, these candy legos. :)

Natalie: I'm excited and impressed by these conversations. I think these questions are big and hard to grapple with. Some of it is terminology, chassis vs genome vs synthetic genome. And how what we're doing differs from what we've done for 30 years. We can cite examples of breakthroughs, but it is a continuum. There is modification in natural cells that we've been doing for 30 years with rDNA technology. E. coli were never meant to make insulin. In that sense they are synthetic. But we're talking engineering of cells and it seems to have turned a corner. I'm glad they spent time on Venter because it really did turn a corner. We are not yet at a place were you can type in a genome and spit it out and boot up a computerlike cell with it. They had to use yeast to stitch together the sequences. But the synthetic piece of DNA is now in a living cell.

John: The synthetic sequences have been inserted into bacteria and yeast?

Natalie: Yeah. It depends where you want to parse it. There are complex programs you could use to make mammalian cells produce cartilage. Those are complex pieces of synthetic DNA. I think the longest piece of synthetic DNA is the venter piece, but that takes an enormous institute to accomplish it. It was a technical tour de force. Right now people are assembling a few thousand BP together to make simple circuits like the banana smell. The difference between Venter and banana smell is that our cells weren't smelling like banana naturally. Venter is calling it a synthetic cell but the sequence is based on what nature gave him, with a few watermarks. So the words are tricky! And there's a continuum of progress here and words get used in different ways and it gets to be complicated.

John: So GMO corn, recombination, they've taken DNA from another organism and put it in corn.

Natalie: Think back to the dead houseplant organism example... The thing with inserting camel and chameleon genes would be a traditional genetic engineering approach. But if you're talking about large scale redesigning of an organism...

Jen: Talking about scale.

Natalie: Yeah. From making a tolerant houseplant to growing a house from an acorn, we need new tools.

Marianne: What does "boot up" mean?

Natalie: It means to start up. If you put in an new OS on your computer it's taking that information and starting up again. To boot up a cell you remove the genetic content and put in a new genome and start it from scratch. Venter didn't do that either. He put a new genome in and waited for it to divide and picked the one that had only the synthetic genome in it. Calling it "the first synthetic cell" is a very far reach.

Lisa: What's the advantage of doing this?

Natalie: When you work with natural stuff, it isn't organized. If we could build an organism and knew everything about it, it would be human understandable. Existing genomes don't come with manuals or organization.

John: We used to crossbreed crops, now we can modify and shuffle genes. Then we were genetically engineering too!

Jen: To eliminate unpredictability. There could be some random enzyme cascade in an organism that you don't know about.

Lisa: And with all their expertise it took one base to screw them up.

Marianne: "You don't know it till you can teach it". Is that like "you don't know it until you can build it"?

Natalie: You and Richard Feynman both think that. If I can't build it I don't understand it. As a scientist, if you build something and it doesn't work, that's informative. There's a nice positive feedback where sci and eng help each other.

Lisa: And even despite all your expertise, clerical errors and accuracy are important.

Natalie: The error could have come in at many points. Some mutation or frameshift... An interesting direction to go. The notion of predictability is at the heart of this next lab. We take a single genetic program and have to know that one cell is not the same as another cell. We call it just a cell... We're going to compare the behavior of the same program in different chassis. To make this truly engineering you want preditable behavior. Biology isn't like that yet.
--

Anne and Anndy present Intro to Color Lab

Now we're going to put a device in a chasis. In fact 2 chassis. This is from the 2009 Cambridge iGEM project.

First define a problem. Toxins contaminate the environment. Detection is expensive and complicated. Can we use bacterial sensors?

They knew that there are natural colorful bacteria and the color genes could be gotten to make purple and green in E. coli.

If all five genes in the ORF are expressed you get purple. If you take out the third one you get green.

Your chassis is E. coli. Two strains, 4-1 and 4-2.

Two plasmids, pPRL and pGRN.

So we want these to work at the maximal level to produce color. Does our device work better in 4-1 or 4-2, or does it not matter? That's our question.

Transformation details. Whenever you transform a bacteria you have to select them. One common way is to plate them on a plate that selects that organism. In most cases you use ampicillin as a selecting agent. That antibiotic is in the media. When you put the plasmid in the bacteria you're also putting the plasmid that codes for Amp resistance in as well. If the E. coli takes up the plasmid it also confers resistance to ampicillin and you see colonies. If it has not taken it up it will not grow.

Lisa: Is there a certain % of cells that take up the plasmid?

Anne: You'll find out!

Anndy: It's like a survey. An aliquot of cells is like a survey of cells. So you'll have these rectangles of cells and eppendorf tubes. Aliquot the plasmids. Each pair of people will use either cell line 4-1 or 4-2. Mix the cells with the plasmid and put it on ice. You have a bunch of plates, some with Amp. Take the cells and heat shock them in a temperature block. Heating them up makes the DNA go in. Then you put them on ice and the cell walls get fixed. Then add Luria broth, "chicken soup for cells", into the mixture, so you can plate them on the plates. Did you plate cells earlier? No? We'll plate some cells. Then let it dry a little and turn them upside down and overnight the cells will grow. Then you see colonies the next day. You have extra plates so you run a + and - control. What might be a good + control? - control? Think about ampicillin resistance.

John: Plate out an aliquot of cells if you haven't added any plasmid so you know they're not Amp resistant.

Anndy: How would you know?

John: Culture the cells on an Amp plate. If they're not resistant they won't grow. And you want that result.

Anndy: That's negative, what do you want for postive?

Lisa: To see the cells grow at all, plate them with no Amp.

Anndy: Yes. Each group of 4 will have a bunch of plates.

Anndy: The stuff [I missed a few minutes of procedure explanation while trying to sort out plates.]

Anne: The biggest drawback is you have to be good with a micropipettor. Before I start with my students I have them practice. Also timing. Those are the two things that can be a problem in this lab.

Anndy: Repeat procedure with pGRN, don't make duplicate controls. Label everything. Think twice before you pipet. Use ice.