1 / 16

“Viral Smart Bombs”

“Viral Smart Bombs”. Joe Levine April 27, 2007 Caltech iGEM Team Brainstorm. Outline. Why viruses for iGEM? Project outline: targeting viruses to specific cells. Technical point: An in vivo aptamer selection scheme to increase specificity. Viruses?. Pro:

lita
Download Presentation

“Viral Smart Bombs”

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. “Viral Smart Bombs” Joe Levine April 27, 2007 Caltech iGEM Team Brainstorm

  2. Outline • Why viruses for iGEM? • Project outline: targeting viruses to specific cells. • Technical point: An in vivo aptamer selection scheme to increase specificity.

  3. Viruses? • Pro: • Certain systems (l, M13, T7) are well understood. • Both molecular biology and biochemistry well characterized. • Wide variety of useful mutants available. • Short life cycle, short iteration times? • Even used for undergraduate lab classes (e.g. MIT 20.109, http://openwetware.org/wiki/20.109) • Con: • Hard to visualize • Usually assayed through interaction with other organisms • Is there an interested resident faculty virus expert? • Opinion: The relative ease and speed of viral manipulations may fit the limitations of iGEM, but lack of local expertise is a real worry.

  4. Virus Life Cycle

  5. Outline • Why viruses for iGEM? • Project outline: targeting viruses to specific cells. • Technical point: A completely in vivo aptamer selection scheme to increase specificity.

  6. Project Overview: Targeting Viruses Specifically & Synthetically • Can we engineer viruses to target cells expressing specific proteins or mRNA’s? • mRNA’s: easier? • Proteins: foreign proteins, or post translationally modified (phosphorylated, prions, etc)? • Virus infects and lyses cells containing target molecule. • A construct in the virus prevents it from killing other cells.

  7. Nucleic Acid Based Sensors? B Target mRNA A B A* (RBS) • Detecting mRNA’s: • Stem loop invasion • Tuneable specificity • Designable1? • Detecting other molecules: • Aptamers2,3,etc • Protein conformers, foreign molecules, etc. • In vivo functioning in the presence of confounding molecules may require in vivo selection B*/2 A* A* B*/2 A A* (RBS) Viral mRNA Covalent modification Prions • Isaacs et al. 2004. • Ellington and Szostak, 1990. • Bayer & Smolke, 2005.

  8. The plan and system • E. coli makes a sensible initial host cell • Well studied phages: l, T7, M13 naturally target E. Coli • Easy to induce varying expression of heterologous target. • Picking targets of increasing difficulties: • 1st stage: A well defined mRNA (GN?) • 2nd stage: A heterologous mRNA • 3rd stage: An easy heterologous protein (lysozyme?) • 4th stage: A difficult protein (phosphorylation state of two component response regulator?) • Hopefully goals #1, and maybe #2, achievable. #3 would be outstanding. I would be absolutely shocked to see #4 over the summer.

  9. What specific genes to block? • Early viral life cycle genes • The antiterminator ‘N’ in l • DN virus strains do not infect1 • Other strains (M13, T7) might have similar candidates • These should prevent the virus from beginning its developmental life cycle • Maybe target aptamers to later stage genes, to allow aptamers time to mature

  10. Possible problems • Proteins may be a challenging substrate for aptamers to recognize. • Specificity (false positives) will be a major issue. • How will the devices work with the virus? • Can we address these problems with in-vivo selection schemes?

  11. Selecting aptamers in vivo • Aptamers are typically selected using in vitro assays. • These selection methods do not mimic in vivo constraints: • Lots of background molecules • Unsure if random proteins will trigger aptamers. • A selection scheme that mimics cellular environment might help.

  12. 8 8 8 “In Vivo SELEX” – Positive Selection • Important caveat: Nvirus << Ncell, else cells will get infected by multiple viruses and unsuccessful aptamers will piggyback on successful ones. Lysis by successful infectors 8 8 8 Infect 8 8 8 8 Purify phage, repeat if desired.

  13. 8 8 8 8 8 8 8 “In Vivo SELEX” – Negative Selection • Want to select for viruses that do not infect cells. This is hard. • Penalize aptamers opening in the absence of ligand. Gene remains repressed Lysis N Repression non-specifically relieved N N Promoter Counterselection kills cells Counterselection N Repressing structure E. Coli counterselection: sacB (sucrose sensitivity), rpsL (streptomycin sensitivity)?

  14. % selectsim.m % % selection dynamics. % Nij is a vector of the number of viruses that are of type ij, % with: % i: 0/1 if aptamers don't/do open in the present of ligand % j: 0/1 if aptamer don't/do open in the absence of ligand % % Mijk is a number describing the multiplier that virus type ij goes % through on round k. ij are the same indices as above, k is 0 for negative % selection and 1 for positive selection. Nrounds = 100; Ratio = 5; M000 = Ratio; % aptamers not opening in either case multiply during negative selection. M001 = 1; % aptamers not opening in either case do not multiply during positive selection. M010 = 1; % aptamers opening only in the absence of ligand do not multiply during negative selection. M011 = 1; % aptamers opening only in the absence of ligand do not multiply during positive selection. M100 = Ratio; % aptamers opening only in the presence of ligand multiply during negative selection. M101 = Ratio; % aptamers opening only in the presence of ligand multiply during positve selection. M110 = 1; % aptamers opening all the time do not multiply during negative selection. M111 = Ratio; % aptamers opening all the time do multiply during positive selection. N00 = zeros(1,Nrounds); N00(1) = 0.33; N01 = zeros(1,Nrounds); N01(1) = 0.33; N10 = zeros(1,Nrounds); N10(1) = 0.01; N11 = zeros(1,Nrounds); N11(1) = 0.33; for j = 2 : Nrounds if mod(j,2) % even round = negative selection N00_new = M000*N00(j-1); N01_new = M010*N01(j-1); N10_new = M100*N10(j-1); N11_new = M110*N11(j-1); total = N00_new + N01_new + N10_new + N11_new; N00(j) = N00_new/total; N01(j) = N01_new/total; N10(j) = N10_new/total; N11(j) = N11_new/total; else N00_new = M001*N00(j-1); N01_new = M011*N01(j-1); N10_new = M101*N10(j-1); N11_new = M111*N11(j-1); total = N00_new + N01_new + N10_new + N11_new; N00(j) = N00_new/total; N01(j) = N01_new/total; N10(j) = N10_new/total; N11(j) = N11_new/total; end end plot(1:Nrounds,N00,1:Nrounds,N01,1:Nrounds,N10,1:Nrounds,N11) legend('N_{00}','N_{01}','N_{10}','N_{11}')

  15. Sketch of selection dynamics? Populations oscillate on alternate rounds due to alternating selection types.

  16. Lots of Details to Work Out!!! • Which virus to use • Culture conditions • How exactly to clone into them. • Etc…? • Thanks for your attention!

More Related