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| ==<center>Project Overview</center>== | | ==<center>Project Background</center>== |
| The Caltech iGEM 2007 team is composed of four undergraduates from Caltech and one undergraduate from MIT. Team members are current juniors and seniors in biology, chemistry, chemical engineering, and biological engineering. The team was advised by three graduate students and three faculty mentors.
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| Our project tries to answer the following question: can viruses be engineered to selectively kill and/or integrate into specific subpopulations of target cells, based on their RNA or protein expression profiles? This addresses an important issue in gene therapy, where viruses engineered for fine target discrimination would selectively kill only those cells over or underexpressing certain disease or cancer associated genes. An even more ambitious goal would be to <i>rewire</i> target cells, by integrating a small gene cassette which would modify the target cell's expression profile, possibly fixing a disease state.
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| While this is clearly an ambitious goal, we managed to choose a simple model system for this problem suitable for undergraduates working over a summer. The bacteriophage λ is a classic, well studied virus capable of infecting E. coli, another classic model genetic sytem. We therefore seek to engineer a λ strain targeted to lyse specific subpopulations of ''E. coli'' based on their transcriptional profiles. Together, λ and E. coli provide a tractable genetic system for this larger problem, while hopefully providing lessons applicable to more ambitious, future projects.
| | [[IGEM:Caltech/2007/Project/phage|Introduction to Bacteriophage λ]] |
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| | [[IGEM:Caltech/2007/Project/Working with Lambda|Working with λ]] <b>(-can someone type up this page, most likely pradeep as this is your main project goal. Brief discussion of amber mutants/suppressors, titering)</b> |
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| Briefly, our project relies on controlling key viral developmental processes in a target-cell specific manner. In our design, the engineered viruses are capable of entering all cells. The viruses are engineered to lack the native copy of a key developmental gene, while containing a second, regulated, copy which is only expressed when the virus infects specific target cells. Thus, viruses infecting non-target cells stall early in their development and are quickly destroyed by the host. Viruses infecting target cells, however, manage to express these essential genes and successfully complete their infection cycle.
| | ==<center>Project Details</center>== |
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| As an initial mechanism to target viruses to specific cell types, we will place the viral developmental genes under riboregulator control. Viral mRNAs for the regulated developmental genes will express with a stem loop sequestering ribosome binding sites, preventing translation. Specific mRNA in target E. coli will invade the stem loop, freeing the ribosome binding site and allowing proper translation. We believe this approach is more general than methods which might target specific cell-surface markers. Furthermore, if this method works, it would be possible in principle to extend viral mRNA regulation using aptamers capable of recognizing subtle signals such as post-translational modification.
| | The project consisted of five individual parts, each assigned to one team member, and categorized into one of three independent principles underlying the project: |
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| We selected the viral developmental genes <i>N, Q,</i> and <i>cro</i> as promising targets for regulation. <i>N</i> and <i>Q</i> are <i>antiterminators</i> required for λ to transcribe its full set of genes. Viruses lacking these genes stall at extremely early developmental stages and are completely inviable, barely producing any viral mRNA. <i>cro</i> represents a potentially interesting means to bias whether the virus will lyse a target cell, or integrate into its DNA. This makes it an attractive candidate to investigate the rewiring applications mentioned above.
| | * [[IGEM:Caltech/2007/Project/Recombineering|Recombineering]] the cro, N, and Q amber mutation genes into lambda zap |
| | * [[IGEM:Caltech/2007/Project/Riboregulator|Riboregulator]] design and testing |
| | *Titering [[IGEM:Caltech/2007/Project/Cro|Cro]], [[IGEM:Caltech/2007/Project/N|N-antiterminator]], and [[IGEM:Caltech/2007/Project/Q|Q-antiterminator]] construct-containing bacterial to test construct-mediated rescue of lytic activity (in amber phage) |
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| Choosing an appropriate λ strain poses a challenge. Existing strains with defective <i>N, Q,</i> and <i>cro</i> genes lack unique restriction sites to clone our constructs into. Therefore, we will utilize recombineering to introduce introduce these mutations into phages specifically designed to accept cloning inserts.
| | ==<center>Current Status</center>== |
| | As the recombineering, testing of riboregulators, and titering processes will take place concurrently, we needed to find a simpler way to regulate viral protein concentrations in the cells. To this end, ''E. coli'' strains have been constructed that contain a low-copy plasmid construct where one of three key developmental viral genes - coding for the cro, N, or Q proteins - is regulated by a tetracycline-dependent promoter. A constitutive promoter (J23100, the stronger promoter, or J23116, the weaker one) produces a steady stream of tetracycline repressor (tetR), which substitutes for the cis repressor in repressing protein levels. The addition of anhydrotetracycline (aTc, acting as the trans activator) inactivates the tetracycline repressor and leads to the production of the respective viral protein in the ''E. coli'' cells. This allows us to control the concentration of viral protein produced in the cells by adding varying amounts of aTc to the bacterial growth media. |
| | * [[IGEM:Caltech/2007/Project/construct|Explanation of Construct]] |
| | * [[IGEM:Caltech/2007/Project/constructList|List of Cloned Constructs]] |
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| Working with lambda phage has been new and fun for all of us. We hope you take the time to browse our wiki and learn more about our work!
| | Titering experiments where cro, N, and Q amber phages were allowed to infect D1210 cells containing the built construct show that heterologous N and Q can complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q even further, as it eliminated lysis completely. We were unable to express sufficient cro from a plasmid to rescue lytic behavior of the amber cro mutant phage. |
| | * [[IGEM:Caltech/2007/Project/titerResults|Summary of Titering Results]] |
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| [[Image:Caltech_2007_overview.gif|center]] | | Multiple riboregulator designs are being tested (for both activation and repression levels), and successful designs will be cloned into the plasmid constructs. So far, cis construct number 3 and its accompanying trans combinations (cis3trans1 and cis3trans2) seem the most promising. Phages resulting from the recombineering process are also being screened for successful N and Q amber mutants. |
| | * [[IGEM:Caltech/2007/Project/cisTransResults|''Trans'' Activation Data]] |
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| [[IGEM:Caltech/2007/Project/phage|Introduction to Bacteriophage λ]]
| | ==<center>Future Work</center>== |
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| ==<center>Project Details</center>==
| | The future of the project lies in confirming trans activation: that is, to prove protein concentration is several times greater in aTc-saturated trans-containing cells as compared to aTc-saturated strains with no trans plasmid. The successful complementary cis-trans pairs can then be incorporated into the N-J23100 and Q-J23116 constructs. Cis repression in the N-construct has yet to be tested, but Q’s results would imply successful repression of lytic action. Based on the presence of plaques on D1210-N and D1210-Q strains even without aTc, even a small amount of trans-activation woudl allow amber phages to successfully enter the lytic cycle. |
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| The project consisted of five individual parts, each assigned to one team member, and categorized into one of three independent principles underlying the project: | | Cis repression of the N and Q constructs can be tested by further titering. A quantitative measure of the increase in protein concentration upon trans-activation will be obtained by fluorescence measurements by flow cytometry. So far, trans activation has been shown in YFP Quanta experiments (cis3-trans1 and cis3-trans2 seem the most promising), although combinations remain that need to be tested. The ultimate test, though, lies in titering amber mutant phage into strains containing both cis and trans plasmids. A positive result (no plaques on cis containing strains, plaques on cis-trans strains) would show successful integration of two independent project components (N/Q protein dependent lysis switch, and the “lock and key” riboregulator). Successful integration demonstrates that standardization on multiple levels (BioBrick parts making each construct, and the more abstract merging of riboregulators into viral decision-making) can allow rapid construction of complex synthetic biological control pathways. |
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| * [[IGEM:Caltech/2007/Project/Recombineering|Recombineering]] the cro, N, and Q amber mutation genes into lambda zap
| | Our current top priority is now to transform the trans1 and trans2 plasmids into the cis3-Q construct cell, titer and check for the presence of plaques. Once successful, we can clone the construct entirely into the recombineered amber phage. In the final system, infecting amber λ-Zap virus will selectively lyse those cells that express the specific trans RNA in their transcriptional profile. |
| *Design and testing of the [[IGEM:Caltech/2007/Project/Riboregulator|Riboregulator]]
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| *Titration curves for [[IGEM:Caltech/2007/Project/Cro|Cro]], [[IGEM:Caltech/2007/Project/N|N-antiterminator]], and [[IGEM:Caltech/2007/Project/Q|Q-antiterminator]] to find out the percentage of bacterial lysis at different concentrations of aTc
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| ==<center>Current Status</center>==
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| Currently, ''E. coli'' strains have been constructed that contain a low-copy plasmid construct where one of three key developmental viral genes - coding for the Cro, N, or Q proteins - is regulated by a tetracycline-dependent promoter. The addition of anhydrotetracycline (aTc) inactivates the tetracycline repressor and leads to the production of the respective viral protein in the E. coli cells. This allows us to control the concentration of viral protein produced in the cells by adding varying amounts of aTc to the bacterial growth media. Heterologous N and Q have been shown to complement phages with amber mutations in the respective genes. Adding a cis-repressor to the Q construct lowered production of Q and prevented complementation. We were unable to express sufficient cro from a plasmid to rescue a cro mutant phage.
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| Multiple riboregulator designs are being tested (for both activation and repression levels), and successful designs will be cloned into the plasmid constructs. Phages resulting from the recombineering process are also being screened for successful N and Q amber mutants.
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| |} | | |} |
| </div> | | </div> |
