IGEM:MIT/2008/Brainstorming Old

Engineering Yogurt Bacteria

about the p1025 peptide

PMID 9920267: A synthetic peptide adhesion epitope as a novel antimicrobial agent.

about yogurt bacteria

Wikipedia's page: What's in yogurt?

Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus lactis

PMID 11772607: electro-transformation and several useful plasmids described
PMID 12788739: a secretion signal sequence described
Another integration plasmid used for transformation of Lactobacillus

adhesion assay for binding of S. mutans to hydroxyapatite

These are old protocols. We'll contact researchers at Forsyth Institute to find a good protocol.
PMID 6325348 and PMID 17045503: an adhesion assay to quantify association between oral Streptococcus pathogens and saliva-coated hydroxyapatite beads

1. • For help setting up a team lab notebook, please contact User: Ricardo Vidal. He's working with OpenWetWare at MIT this year, and could help you all get familiar with some new tools that update the notebook since last year's iGEM notebooks. - Jason R. Kelly
2. Should we schedule a regular meeting? Chia: Feedback from last year's advisers: regular meetings among grads are recommended. During summer, weekly lab meetings with the undergrads would be important. Perhaps let them rotate to be the team leader and lab meeting presenter so everyone gets a chance to practice their leadership skills.
3. iGEM requirements and relevant dates:
   • Team registration deadline is May 9; need to specify instructors, students, lab space, and funding. Registration fee $1000/team.
   • Jamboree: November 8-9, 2008 at MIT; $100 per undergrad and $250 for others.

Brainstorming Project Ideas

iGEM research project tracks:

1. Foundational Research - basic science and engineering research
2. Information Processing - genetically encoded control, logic, and memory
3. Energy - biological fuels, feedstocks, and other energy projects
4. Environment - sensing or remediation of environmental state
5. Health & Medicine - applied projects with the goal of directly improving the human condition
6. (new track this year) - software tools to facilitate use of standard biological parts

Information Storage Device

I was thinking a bit about information storage. There have been a whole slew of papers that suggest storing artificial messages in DNA. A few representative examples are

• C. Bancroft, T. Bowler, B. Bloom, and C. T. Clelland, "Long-Term Storage of Information in DNA," Science, vol. 293, no. 5536, pp. 1763-1765, Sept. 2001.

• P. C. Wong, K.-K. Wong, and H. Foote, "Organic data memory using the DNA approach," Commun. ACM, vol. 46, no. 1, pp. 95–98, Jan. 2003.

• J. P. L. Cox, "Long-term data storage in DNA," Trends in Biotechnology, vol. 19, no. 7, pp. 247-250, July 2001.

All of these works envision long-term storage where complicated cloning techniques with restriction enzymes, oligonucleotide synthesis or PCR, and ligation would be used for storage. I don't know much at all about this, but are there ways of making a storage device where it is moderately easy to change what is written in the memory? Basically designing some kind of encoder and decoder that makes DNA more of a rewritable medium rather than just a long-term storage medium. A related question is whether there might be a way to introduce an error-correcting circuit along the lines of

• M. G. Taylor, "Reliable information storage in memories designed from unreliable components," Bell Syst. Tech. J., vol. 47, no. 10, pp. 2299–2337, Dec. 1968.

There are probably more easily editable means of biological information storage that are more worthy of exploration.

• cookb: Hmm, that's an interesting idea and quite original for iGEM! I'm getting an image of some sort of simple physical signal (e.g., exposure to light) being converted into DNA information, sort of like a Morse code encoding words into DNA. I wonder what possible mechanisms one could use to facilitate that... But even encoding something simple like "Hello world" would be a huge deal, and have big ramifications on areas such as commercial gene synthesis.

• Use Fosmid to control copy number of device in E coli?

Recombination-based tools to alter DNA sequences

• High frequency deletion of a DNA region: Flank the region with two identical target sequences recognized by a DNA recominase (Cre or Flp) diagram

• Inversion of a DNA region (low reversion rate): Flank the region with two mutant loxP sequences, lox66 and lox71.

• • Cre recombinase-mediated inversion using lox66 and lox71: Method to introduce conditional point mutations into the CREB-binding protein

• • A review article listing WT and mutant target sequences recognized by Cre and Flp. These mutants can be used to create stable recombinant products. See: Road to precision: recombinase-based targeting technologies for genome engineering. PMID:17904350

• • The FLP Recombinase of the Yeast 2-µ m Plasmid: Characterization of Its Recombination Site

• Reversible inversion of a DNA region: Flank with target sequences of the Hin recombinase. See 2006 Davidson team page

• Transposons:
  • Sleeping Beauty (transposon from fish) Molecular Reconstruction of Sleeping Beauty

Write-Once Memory Information Theory

• Lav R. Varshney 23:55, 17 April 2008 (EDT): A couple of basic papers on write-once memories are:

• Ronald L. Rivest and Adi Shamir, "How to Reuse a 'Write-Once' Memory," Inf. Control, vol. 55, no. 1-3, pp. 1-19, Oct.-Dec. 1982.

• Gilles Zemor and Gerard D. Cohen, "Error-correcting WOM-codes," IEEE Trans. Inf. Theory, vol. 37, no. 3, pp. 730-734, May 1991.

• Amos Fiat and Adi Shamir, "Generalized 'write-once' memories," IEEE Trans. Inf. Theory, vol. IT-30, no. 3, pp. 470-480, May 1984.

The following paper that I am working on is also rather related, so if anyone wants to take a look at the unsubmitted draft, let me know:

• Lav R. Varshney and Julius Kusuma, "Malleable Compression," in preparation.

Synthetic Taxis

Develop some sort of Kalman filter-like circuit or some other signal processing circuit to detect or track pathogens. The UCSF/UCB Center for Engineering Cellular Control Systems has started to look at some similar problems.

Bacterial lava lamp

• Reshma 10:16, 19 March 2008 (CDT): I've been wanting to make a bacterial lava lamp for a long time. The U. of Melbourne 2007 team engineered this super cool part that enables bacteria to float (used in natural systems to maintain marine bacteria at a particular depth). By combining this floatation part with a luciferase, I think you could make some nice lighting for the home!  :)

(from Melbourne 07 team) - they just sink more slowly atm ;)

If we did this, we would want the cells to clump together so that it looks lava lamp like.

How is this combo: Flocculating E. coli + increased buoyancy + GFP or luciferase on cell surface + self-made tube-flipping device (mechanical or manual) + blue excitation light or luciferin? A name for this GEM suggested by Brian: E.GloLite

• Display of green fluorescent protein on Escherichia coli cell surface.
  • Jason R. Kelly 16:46, 4 April 2008 (EDT):You probably don't need to get it on the surface, they glow pretty well if you just make a bunch of GFP inside the cell (also easier to do).

• Chia: A microbe-based lava lamp - cute! We can make budding yeast cells clump as well. There is a family of cell surface glyco-proteins that confer cell-cell adhesion (flocculation) and/or adhesion to hydrophobic substratum [1]. Flocculation mediated by the protein Flo1p can be altered by adding certain sugar or EDTA. Yeast cell density can be altered by mutations in secretory pathways such as sec1.

Two scientists made a microbial lava lamp. Not as fancy as what we want here but we might find their design useful. Basically, brewer's yeast cells are immobilized in a mixture of glass beads, a carbohydrate polymer from seaweed and some dye to make colored "beads". Then the beads are placed in a sucrose solution allowing yeast cells to produce CO2, which is trapped in the beads and giving them buoyancy. As the beads slowly rise to the surface, CO2 escapes and the beads drop. PDF describing details

Multi-colored moss

• Jason R. Kelly 12:23, 19 March 2008 (CDT): Austin and I went out and visited Magdalena Bezanilla's lab at Umass-Amhearst that studies this moss and found out that it's not too hard to grow and genetically manipulate it. One idea was to express pigments from other plants (flowers) in the moss to make it in different colors. Magdalena thought this might be possible, could follow up with her if it was an idea that folks thought was cool. Even if it didn't work, you could make the first BioBrick parts and vectors for engineering plants!

• The moss (Physcomitrella patens) just had its genome sequenced![2]
• P. patens lends itself well to homologous recombination (>50%/mcg DNA), and we should be able to transform a stable gene into it quickly and easily.
• RNAi works handily with the P. patens, too. Japanese scientists a while ago made the first blue rose using RNAi coupled with some metabolic engineering: [3]. Perhaps we can make the first genetically-engineered blue moss?
• This project is probably going to be metabolic engineering. In order to get some kind of blue or red color into the moss, we would probably try to make one of the anthocyanins using endogenous and heterologous enzymes in the moss. Anthocyanins [4] are the pigments that make flowers and fruits blue (delphinin, a common anthocyanin, was used to make the blue rose). Recently, people engineered anthocyanin production into E. coli [5], so it seems that it should be easy in a plant, considering the moss likely already has many of the endogenous enzymes to make necessary precursors.
• What's nice about this project as well is that most of the work to build the vectors and genes we want will have to be in E. coli. So, while we're waiting for the moss to grow, we can stay busy making biobrick vectors for P. patens, which would be very useful to any and all P. patens labs out there.
• A very useful review on plant pigment metabolism [[6]]. Anthocyanin metabolism is well understood and has been the focus of much biotechnology lately. Some anthocyanins are also important in human diet as antioxidants. The flower market is ~70$billion/year! Modifying flower colors/appearance/longevity is big business!

Engineering Yogurt Bacteria

A study on the short peptide QGRVEVLYRGSWGTVC that competes for binding to tooth surface with Streptococcus mutans Reference What's in yogurt?

Streptococcus thermophilus
Genome sequence browser
PMID 14532023: food-grade cloning vector and industrial strains

Lactobacillus delbrueckii subsp. bulgaricus
PMID 11772607: electro-transformation and several useful plasmids described
PMID 12788739: a putative cell wall localization signal described
Another integration plasmid used for transformation of Lactobacillus
PMID 6325348 and PMID 17045503: an adhesion assay to quantify association between bacteria and saliva-coated hydroxyapatite beads

Bacteria with limited # of cell divisions, after flipping a switch

A neat idea initiated by Vikram. Such a GEM would have much less potential to contaminate the environment if accidentally released from the laboratory.

This might be possible if the bacterial genome shortens gradually like linear eukaryotic chromosomes. A method to linearize the E. coli genome without affecting its stability has been reported [7].

If the chromosome ends were generated differently, one might be able to implement a replication dependent shortening mechanism. (Brain power needed!)

One way to visualize chromosome shortening (besides PCR or Southern blotting) could be insertion of a reporter at various locations of the linearized chromosome. Ideally the presence/absence of the reporter would make E. coli colonies look different on a agar plate. (Say lacZ + X-gal as in blue/white screen?) If the reporter is lost right after the first division (since it's inserted very close to the initial chromosome end), the colony would be one color. If the reporter is inserted far away from chromosome ends and thus retained for many generations, the colony would be another color. - Chia

Other ideas from dinner meeting on 3/31

(feel free to edit!)

1. modulation of heterocyst differentiation. See Heterocyst Differentiation and H2 Production in N2-Fixing Cyanobacteria
2. creative use of FLP recombinase for storing/removing DNA based information
3. modulation of com sensing in B. subtilis (model system in Alan Grossman's lab in the Bio Dept)
4. a bacterial diagnostic device for diabetes?

Suggestions from the meeting on 4/4

1. Various people are going to do background research on technical aspects of ideas listed above. They plan to report back on 4/11.
2. Additional idea of making bacteria responsive to electrical stimuli.

Suggestions from the meeting on 4/17

• cookb: Use of inteins (protein introns) as switches / biosensors

1. Big advantage is their modular design which allows easy creation of new functional systems
2. Could use to switch on reporter/effector using temperature change or addition of ligand
3. Huge possibilities as a biosensor (pop in ligand binding domain of choice)
   • Some ideas: glucose sensor (diabetes), pollutant detector (metal binding domains), cancer test (VEGF, other GFs), in vivo phosphotyrosine assay (SH2 domain), detection of biohazards/pesticides/organophosphate, drug testing (steroids)...

Relevant papers: (links haven't been updated for the new wiki layout)

1. Skretas G and Wood DW. Regulation of protein activity with small-molecule-controlled inteins. Protein Sci. 2005 Feb;14(2):523-32. DOI:10.1110/ps.04996905 |
2. Skretas G and Wood DW. A bacterial biosensor of endocrine modulators. J Mol Biol. 2005 Jun 10;349(3):464-74. DOI:10.1016/j.jmb.2005.04.009 |
3. Gillies AR, Skretas G, and Wood DW. Engineered systems for detection and discovery of nuclear hormone-like compounds. Biotechnol Prog. 2008 Jan-Feb;24(1):8-16. DOI:10.1021/bp070144i |
4. Skretas G, Meligova AK, Villalonga-Barber C, Mitsiou DJ, Alexis MN, Micha-Screttas M, Steele BR, Screttas CG, and Wood DW. Engineered chimeric enzymes as tools for drug discovery: generating reliable bacterial screens for the detection, discovery, and assessment of estrogen receptor modulators. J Am Chem Soc. 2007 Jul 11;129(27):8443-57. DOI:10.1021/ja067754j |
5. Zeidler MP, Tan C, Bellaiche Y, Cherry S, Häder S, Gayko U, and Perrimon N. Temperature-sensitive control of protein activity by conditionally splicing inteins. Nat Biotechnol. 2004 Jul;22(7):871-6. DOI:10.1038/nbt979 |
6. Buskirk AR, Ong YC, Gartner ZJ, and Liu DR. Directed evolution of ligand dependence: small-molecule-activated protein splicing. Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10505-10. DOI:10.1073/pnas.0402762101 |
7. Ostermeier M. Engineering allosteric protein switches by domain insertion. Protein Eng Des Sel. 2005 Aug;18(8):359-64. DOI:10.1093/protein/gzi048 |

Suggestions from the meeting on 4/24

• Fundraising! Potential donors = MIT alumni (Need to create an official web site, a brochure and a standard letter/budget for continuous fundraising for MIT's iGEM team every year
• Meeting registration deadline = May 9th
• Combinatorial use of Flp and Cre target sites (see "Recombination-based tools to alter DNA sequences" above) to store info in DNA
• Couple recombinases to inteins to make novel a sensor/pathway
• Talk to Prof. Penny Chisholm to learn the basics of handling Cynanobacteria. (Growth condition and doubling time? Any potential toxicity?)

Notes from 5/1/

Still brainstorming:

1. still thinking about computation in cells
2. brian mentioned something about bacterial films on our teeth

• yeast that makes a peptide that prevents bad streptococcus from binding from teeth.

1. something for sensing
2. phosphotyrosine sensing (SH2 domain) coupled to

Notes from 5/28

Goal for today: present project ideas to drew and tom and help us narrow it down to three ideas

We should have a grad student advisor on site at all times. Felix will be in lab all the time. Do we have an out of lab spot (lunch room is a good fall back). We should set a meeting for logistics and operations for next summer.

Felix - metabolic engineering of moss physcomitrella patens for pigments production. make different plant pigments in mosses. advantages - will be making plant hacking biobrick parts. haploid most of the time. easy to knock out genes. transformable with PEG and high frequency of homologous recombination. need some equipment. graham walker's lab may have the equipment that we need to grow the moss. What would we want to do with them if we could engineer them? If you pick color, could you speed this up and do construction and testing in E. coli or yeast? You could put two promoters in front of it and have one work in e coli and one in moss. How much engineering is involved? Lots of foundational stuff would come out of the project

Brian - Intein based biosensor when ligand binding domain binds ligand, intein splices itself out and reporter protein gets turned on. previous work is on hormone detection (estrogen receptor). could we use this for glucose (from glucokinase) for diabetes, phosphotyrosine (from SH2 domain) live cell imaging, VEGF, metal-ions, pesticides. Maybe could try multiple ligand binding domains and intein parts would be great for registry. cons = functionality might require directed evolution, intein switch is not reversible.

Chia - surface expression / secretion of a synthetic peptide by lactobacillus bulgaricus in yogurt bacteria Streptococcus mutans causes cavities. SA I/II is important for adhesion to tooth surface via a glycoprotein in our mouths. SM changes pH wchich causes damage to teeth. there is a 20 amino acid peptide that can block binding to teeth. a clinical trial had people use this peptide for 120 days and compared to control peptide and no peptide. reduced the amount of SM on teeth and in saliva. make sure that this peptide will be secreted not surface expressed. is there an antibody to the peptide? look at binding to hydroxyapatite beads. Tom - problem is that this would leave us with a strain that was antibiotic resistant, make sure we don't end up with that.

Chia - controlled heterocysts formation to control for the right percentage of heterocysts in order to maximize for nitrogen fixation. is there a local expert? no, but bob at uchicago is the expert- he would be excited to have anybody working on this. Drew feels that we are missing the cool application for this

Scott - make recombinase operator sites that let us control the recombinase locations. you'd get a multi-input DNA writing. if you put one repressor on each end of the recombinase site, you get some combinatorial effect. Comments: how much leakage would you get? how much does this overlap with the pancake flipping

Brian - autoprepping bacteria clumping of cells with leucine zippers on cell surface, cell lysis with lysozyme, degrade RNA with RNAse A.

Drew liked the moss, the autoprepping bacteria, and the yogurt ideas because they each have their own applications and that will help us hit three separate application areas that the students will be interested in. We should pick three (not necessarily these three ideas) and be ready to present them.

We'll meet next Wednesday evening - same time and place