IGEM:Caltech/2007/Project

From OpenWetWare

< IGEM:Caltech | 2007(Difference between revisions)
Jump to: navigation, search
Current revision (14:29, 26 October 2007) (view source)
(<center>Project Details</center>)
 
(35 intermediate revisions not shown.)
Line 9: Line 9:
|-valign="top"
|-valign="top"
|style="background:#ffffff"|
|style="background:#ffffff"|
-
==<center>Project Overview</center>==
+
==<center>Project Background</center>==
-
The Caltech iGEM 2007 team is composed of four Caltech undergraduates and one undergraduate from MIT. Team members are current juniors and seniors in biology, chemistry, chemical engineering, and biological engineering. In addition, three graduate student advisors, as well as three faculty advisors are involved in helping the progress of the team's work.
+
-
The goal of our project is to create a λ bacteriophage that can selectively lyse specific subpopulations of ''E. coli''. We will first use recombineering techniques to insert amber mutations into three key developmental genes in λ-Zap. Next, a second copy of these genes, controlled by a cis-repressing riboregulator, will be cloned into the phage genome. The expression of trans-activating RNA in the target bacterial host will relieve repression, complement the engineered phage, and allow lysis. Hosts which do not contain this RNA will remain intact.
+
[[IGEM:Caltech/2007/Project/phage|Introduction to Bacteriophage λ]]
 +
<br>
 +
[[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>
 +
==<center>Project Details</center>==
-
Currently, ''E. coli'' strains have been constructed that contain a 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. We were unable to express sufficient cro from a plasmid to rescue a cro mutant phage.
+
The project consisted of five individual parts, each assigned to one team member, and categorized into one of three independent principles underlying the project:
 +
* [[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)
-
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.
+
==<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]]
 +
<br>
 +
 
 +
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]]
 +
<br>
 +
 
 +
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]]
 +
<br>
 +
 
 +
==<center>Future Work</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.
 +
 
 +
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.
 +
 
 +
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.
-
==<center>Project Details</center>==
 
-
* [[IGEM"Caltech/2007/Project/Recombineering|Recombineering]]
 
-
* [[IGEM:Caltech/2007/Project/N|N-antiterminator]]
 
-
* [[IGEM:Caltech/2007/Project/Q|Q-antiterminator]]
 
-
* [[IGEM:Caltech/2007/Project/Cro|Cro]]
 
-
* [[IGEM:Caltech/2007/Project/Riboregulator|Riboregulator]]
 
-
==<center>Current Status</center>==
 
-
'''All - where are we now?'''
 
|}
|}
</div>
</div>

Current revision


iGEM 2007

Home        People        Project        Protocols        Notes        Changes       


Project Background

Introduction to Bacteriophage λ
Working with λ (-can someone type up this page, most likely pradeep as this is your main project goal. Brief discussion of amber mutants/suppressors, titering)

Project Details

The project consisted of five individual parts, each assigned to one team member, and categorized into one of three independent principles underlying the project:

Current Status

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.


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.


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.


Future Work

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.

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.

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.


Personal tools