Team:British Columbia/Jammer

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=“Jammer”: Modular Gene Regulation (and Binary Logic) using Reverse Antisense Transcription =
 
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==SECTION 1: STATEMENT OF RESEARCH PROBLEM==
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=Traffic Light Switcher: the Modular and Endogenous Jammer=
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Description of Biological Phenomenon and General Aims of Project
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A fundamental goal of synthetic biology is the construction of modular parts
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==Overview==
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that form genetic circuits, which perform tasks akin to electronic devices.
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Control of these genetic elements is essential for their effective
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performance. Although effective means of controlling gene expression via
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inhibition exist, such methods require well-characterized and effective
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pathways on the part of the biological chassis (e.g. Dicer in RNAi), and
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exogenous amplification are necessary preparation for their use (e.g.
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exogenously produced RNA for siRNA).
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[[Image:E_coli_Traffic_Light_Jammer_General.png|thumb|center|450px|A general explanation of the Jammer component of the ''E. coli'' Traffic Light Biosensor.]]
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==SECTION 2: RESEARCH OBJECTIVES AND SPECIFIC METHODS==
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<BR>
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Here, we aim to develop an endogenous system to control gene expression using
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The traffic light requires a component that switches off a light that was previously turned on. To date, the registry does not have a method to easily turn off genes independently from a forward promoter (e.g. inducible and repressible promoters) or biological chassis (e.g. Dicer). We designed a modular, endogenous method of repression using a reverse antisense promoter. Our proof-of-concept jammer <partinfo>k206010</partinfo> uses pBAD in the reverse direction along with various terminators to knockdown GFP expression.  
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antisense-based inhibition. A BioBrick containing a reverse promoter and
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terminator is assembled downstream of the transcript before its terminator.
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Another terminator is assembled upstream of this transcript, so that the
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completed assembly has a terminator-promoter-transcript-promoter-terminator
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motif.  
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Composite BioBrick parts are assembled using the standard three-antibiotics
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We did the following:
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method. B0014 and J23100/101/105 were assembled as part of the terminator and
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#Assembled a reverse pBAD (<partinfo>J44002</partinfo>) with terminator (<partinfo>B0014</partinfo>) into a GFP generator
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promoter parts respectively (i.e. three strengths of forward constitutive
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#Tested the first version by quantifying GFP expression to determine if knockdown exists
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promoters; inducible forward promoters may enable binary logic if used with an
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#Redeveloped the jammer by adding a forward terminator and optimizing terminator position
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inducible reverse promoter). B0034 and K145015 were assembled as part of the
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#Retested the second version and succeeded
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fast-degrading GFP_LVA reporter transcript. J44002, an arabinose-inducible
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#Submitted BioBrick to Registry
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reverse promoter and B0014 functioned as part of the promoter-terminator.
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The intended conditions of the completed BioBrick part is constitutively
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==Results & Quantification==
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expressed GFP in LB without arabinose. In presence of arabinose, the reverse
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promoter is induced thus reducing the amount of reporter transcript available
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for transcription, leading to reduced levels of GFP. Flow cytometry analysis
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will primarily determine the effectiveness of the device. Results, time and
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material-permitting, quantitative dot blot and RT-PCR may be used for further
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verification of results.
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==SECTION 3: SUMMARY OF RESULTS==
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In our first test, we assembled a constitutively expressed GFP part with the first version of the jammer <partinfo>K206008</partinfo> and quantified using FACS.
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GFP expression will be analyzed by flow cytometry to determine if a detectable
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[[Image:E_coli_Traffic_Light_J23100_Jammer_Incomplete.png|thumb|center|500px|The forward terminator is necessary for proper functioning of the Jammer. Arabinose induction shows no significant effects on GFP expression.]]
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shift exists. Quantitative dot blot analysis may be used for absolute
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quantification. If results can be reproduced, RT-PCR may be used for
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additional verification of the production of an anti-sense RNA transcript.
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The reverse promoter is expected to exert genetic control when induced through
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It appeared our first test did not work as expected. After redeveloping the jammer, by adding terminators and repositioning them, we tested the device again.
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two mechanisms: 1) it recruits RNA polymerase to transcribe in the reverse
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direction, thus colliding polymerases inhibit transcription, and 2) the
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reverse transcript is complementary to the coding transcript, thus they form a
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dsRNA duplex, which solicits anti-sense mediated blocking of the ribosome for
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the translation of RNA and related bacterial degradosome machinery (e.g. RNase
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II, III).
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[[Image:E_coli_Traffic_Light_J23100-Jammer_Complete.png|thumb|center|500px|J23100-Jammer works as expected. Arabinose induces near-total knockdown of GFP expression.]]
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==SECTION 4: SUMMARY OF PROPOSED FUTURE EXPERIMENTS AND TIMELINE OF COMPLETION==
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Here, we have shown that the Jammer works as expected, by inducing a near total knockdown of GFP expression. The forward terminator is necessary for its function, and although the biological mechanism is not clear at this time, we have proposed possible mechanisms below.
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One assembly remains until the final device is complete. Sequence verification
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==Possible Biological Mechanisms of Jammer==
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of the pre-complete assembly should arrive by Monday, Sept. 21. Final assembly
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should be complete by Friday, Sept. 25. Flow cytometry analysis will begin
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immediately and is expected to yield final results by Wednesday, Sept. 30.
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Concurrent quantitative dot blot analysis will be complete by that date, if
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materials exist.
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==Preliminary Results for Immediate Release ­- 11:48pm October 14, 2009 ==
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[[Image:E_coli_Traffic_Light_Jammer_Mechanism.png|thumb|center|500px]]
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* Alex Ng and Amelia Hardjasa
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We proposed that, due to the reliance of a reverse promoter and two flanking terminators in the forward and reverse directions, that the hybridization of sense and anti-sense transcripts are inhibiting GFP expression. It is possible that because the double-stranded RNA is fully complementary, its complementary binding is particularly effective at protecting the transcript from translation. The necessity of the forward terminator suggests that a correctly sized antisense transcript significantly helps knockdown. Another mechanism that is possible suggests that the double-stranded RNA is being targeted for degradation, which would reduce GFP expression. Lastly, the reverse direction of RNA-dependent RNA polymerases may be colliding with the forward direction polymerase that transcribes GFP. However, this mechanism is unlikely to be as strong as the former or other possible explanations because, without the forward terminator, GFP expression is not inhibited.
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==Summary of Results ==
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==BioBrick Submission==
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BW27783 cells containing Jammer plasmids grown overnight show GFP_LVA
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Here, you can find the working jammer <partinfo>K206010</partinfo>. For use by other teams looking for a modular, easily repressible jammer, use <partinfo>K206008</partinfo> along with a forward terminator such as <partinfo>B0014</partinfo>. An experimental weaker jammer is <partinfo>K206011</partinfo>.
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knockdown in the presence of arabinose.  Although these results are very
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preliminary (e.g.  without replicates), they suggest that arabinose is
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inducing the production of an anti-sense transcript that causes knockdown
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of GFP_LVA expression.
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==Methods==
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BW27783 cells with the following plasmids were inoculated and grown
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overnight in 15mL LB with or without 0.5% arabinose: Constitutive GFP_LVA,
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Jammer-100, Jammer-101, Jammer-105, and Empty Cells (no plasmids).  FACS
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data was taken at approximately 18 hours and 24 hours post-inoculation.
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18-hour data is shown.  ODs were taken at 24 hours to verify similar
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grown.  Jammer constructs: Terminator- Constitutive Promoter (J23100
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or J23101 or J23105)-RBS-GFP_LVA-Pbad_reverse- Terminator.  *Note that
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J23100 Jammer has not been sequence verified, only length and fluorescence
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verified.
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==Results==
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<html>
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<img src="https://static.igem.org/mediawiki/2009/3/39/British_Columbia_JammerData_J23100.jpg" height=300>
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</html>
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Astonishingly, Jammer-100 cells grown in presence of arabinose show
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fluorescence almost identical to cells without GFP_LVA plasmids, implying
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near total knockdown levels of GFP_LVA fluorescence.  Note that plasmid
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is not sequence verified; however, failed assemblies should only be
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missing the reverse-strand terminator.
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<html>
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<img src="https://static.igem.org/mediawiki/2009/2/29/British_Columbia_JammerData_J23101.jpg" height=300>
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</html>
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Cells containing Jammer-101 plasmids show 101 knockdown in presense of
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arabinose.  Uninduced populations show two peaks of GFP_LVA expression,
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one at constitutive levels and another at no fluorescence.  It is possible
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that weak transcription of the promoter and rapid LVA degradation tags
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may contribute to non-fluorescent cells.
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<html>
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<img src="https://static.igem.org/mediawiki/2009/0/0f/British_Columbia_JammerData_J23105.jpg" height=300>
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</html>
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Cells containing Jammer-105 plasmids show no apparent GFP_LVA
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fluorescence. It is surprising that arabinose-induced cells have
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measureable fluorescence.
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<html>
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<img src="https://static.igem.org/mediawiki/2009/a/a7/British_Columbia_JammerData_opticaldensity.jpg" height=300>
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</html>
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OD data do not suggest significant differences in viability from potential
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toxicity in LB containing 0.5% arabinose, implying observed knockdowns
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are unlikely to result from dead cells with dysfunctional translational
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machinery for GFP_LVA production.
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==Conclusions==
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FACS data of three independent constructs and ODs taken together strongly
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suggest an observable knockdown of GFP_LVA from arabinose induction.
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Although these results are very preliminary and require additional
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verification, they are exciting results that an endogenous, modular method
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of knockdown at the RNA level is possible, thus potentially enabling
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elegant and rapid constructs of auto-regulation at the RNA-level that
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is independent of the biological chassis.
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Latest revision as of 03:48, 22 October 2009

Contents

Traffic Light Switcher: the Modular and Endogenous Jammer

Overview

A general explanation of the Jammer component of the E. coli Traffic Light Biosensor.


The traffic light requires a component that switches off a light that was previously turned on. To date, the registry does not have a method to easily turn off genes independently from a forward promoter (e.g. inducible and repressible promoters) or biological chassis (e.g. Dicer). We designed a modular, endogenous method of repression using a reverse antisense promoter. Our proof-of-concept jammer uses pBAD in the reverse direction along with various terminators to knockdown GFP expression.

We did the following:

  1. Assembled a reverse pBAD () with terminator () into a GFP generator
  2. Tested the first version by quantifying GFP expression to determine if knockdown exists
  3. Redeveloped the jammer by adding a forward terminator and optimizing terminator position
  4. Retested the second version and succeeded
  5. Submitted BioBrick to Registry

Results & Quantification

In our first test, we assembled a constitutively expressed GFP part with the first version of the jammer and quantified using FACS.

The forward terminator is necessary for proper functioning of the Jammer. Arabinose induction shows no significant effects on GFP expression.

It appeared our first test did not work as expected. After redeveloping the jammer, by adding terminators and repositioning them, we tested the device again.

J23100-Jammer works as expected. Arabinose induces near-total knockdown of GFP expression.

Here, we have shown that the Jammer works as expected, by inducing a near total knockdown of GFP expression. The forward terminator is necessary for its function, and although the biological mechanism is not clear at this time, we have proposed possible mechanisms below.

Possible Biological Mechanisms of Jammer

E coli Traffic Light Jammer Mechanism.png

We proposed that, due to the reliance of a reverse promoter and two flanking terminators in the forward and reverse directions, that the hybridization of sense and anti-sense transcripts are inhibiting GFP expression. It is possible that because the double-stranded RNA is fully complementary, its complementary binding is particularly effective at protecting the transcript from translation. The necessity of the forward terminator suggests that a correctly sized antisense transcript significantly helps knockdown. Another mechanism that is possible suggests that the double-stranded RNA is being targeted for degradation, which would reduce GFP expression. Lastly, the reverse direction of RNA-dependent RNA polymerases may be colliding with the forward direction polymerase that transcribes GFP. However, this mechanism is unlikely to be as strong as the former or other possible explanations because, without the forward terminator, GFP expression is not inhibited.

BioBrick Submission

Here, you can find the working jammer . For use by other teams looking for a modular, easily repressible jammer, use along with a forward terminator such as . An experimental weaker jammer is .

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