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Team:British Columbia/Jammer
From 2009.igem.org
Home Team Traffic Light Sensor Lock&Key Jammer [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=British_Columbia Parts] Safety Sponsors Notebook Bibliography
Traffic Light Switcher: the Modular and Endogenous Jammer
Overview
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:
- Assembled with terminator into a GFP generator
- Tested the first version by quantifying GFP expression to determine if knockdown exists
- Redeveloped the jammer by adding a forward terminator and optimizing terminator position
- Retested the second version and succeeded
- Submitted BioBrick to Registry
Results & Quantification
We assembled
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SECTION 2: RESEARCH OBJECTIVES AND SPECIFIC METHODS
Here, we aim to develop an endogenous system to control gene expression using antisense-based inhibition. A BioBrick containing a reverse promoter and terminator is assembled downstream of the transcript before its terminator. Another terminator is assembled upstream of this transcript, so that the completed assembly has a terminator-promoter-transcript-promoter-terminator motif.
Composite BioBrick parts are assembled using the standard three-antibiotics method. B0014 and J23100/101/105 were assembled as part of the terminator and promoter parts respectively (i.e. three strengths of forward constitutive promoters; inducible forward promoters may enable binary logic if used with an inducible reverse promoter). B0034 and K145015 were assembled as part of the fast-degrading GFP_LVA reporter transcript. J44002, an arabinose-inducible reverse promoter and B0014 functioned as part of the promoter-terminator.
The intended conditions of the completed BioBrick part are constitutively expressed GFP in LB without arabinose. In presence of arabinose, the reverse promoter is induced thus reducing the amount of reporter transcript available for transcription, leading to reduced levels of GFP. Flow cytometry analysis will primarily determine the efficacy of the device. Results, time and material-permitting, quantitative dot blot and RT-PCR may be used for further verification of results.
SECTION 3: SUMMARY OF RESULTS
GFP expression will be analyzed by flow cytometry to determine if a detectable shift exists. Quantitative dot blot analysis may be used for absolute quantification. If results can be reproduced, RT-PCR may be used for additional verification of the production of an anti-sense RNA transcript.
The reverse promoter is expected to exert genetic control when induced through two mechanisms: 1) it recruits RNA polymerase to transcribe in the reverse direction, thus colliding polymerases inhibit transcription, and 2) the reverse transcript is complementary to the coding transcript, thus they form a dsRNA duplex, which solicits anti-sense mediated blocking of the ribosome for the translation of RNA and related bacterial degradosome machinery (e.g. RNase II, III).
SECTION 4: SUMMARY OF PROPOSED FUTURE EXPERIMENTS AND TIMELINE OF COMPLETION
One assembly remains until the final device is complete. Sequence verification of the pre-complete assembly should arrive by Monday, Sept. 21. Final assembly should be complete by Friday, Sept. 25. Flow cytometry analysis will begin immediately and is expected to yield final results by Wednesday, Sept. 30. Concurrent quantitative dot blot analysis will be complete by that date, if materials exist.
Preliminary Results for Immediate Release - 11:48pm October 14, 2009
- Alex Ng and Amelia Hardjasa
Summary of Results
BW27783 cells containing Jammer plasmids grown overnight show GFP_LVA knockdown in the presence of arabinose. Although these results are very preliminary (e.g. without replicates), they suggest that arabinose is inducing the production of an anti-sense transcript that causes knockdown of GFP_LVA expression.
Methods
BW27783 cells with the following plasmids were inoculated and grown overnight in 15mL LB with or without 0.5% arabinose: Constitutive GFP_LVA, Jammer-100, Jammer-101, Jammer-105, and Empty Cells (no plasmids). FACS data was taken at approximately 18 hours and 24 hours post-inoculation. 18-hour data is shown. ODs were taken at 24 hours to verify similar growth. Jammer constructs: Terminator- Constitutive Promoter (J23100 or J23101 or J23105)-RBS-GFP_LVA-Pbad_reverse- Terminator. *Note that J23100 Jammer has not been sequence verified, only length and fluorescence verified.
Results
Astonishingly, Jammer-100 cells grown in presence of arabinose show fluorescence almost identical to cells without GFP_LVA plasmids, implying near total knockdown levels of GFP_LVA fluorescence. Note that the plasmid is not sequence verified; however, failed assemblies should only be missing the reverse-strand terminator.
Cells containing Jammer-101 plasmids show 101 knockdown in the presence of arabinose. Uninduced populations show two peaks of GFP_LVA expression, one at constitutive levels and another at no fluorescence. It is possible that weak transcription of the promoter and rapid LVA degradation tags may contribute to non-fluorescent cells.
Cells containing Jammer-105 plasmids show no apparent GFP_LVA fluorescence. It is surprising that arabinose-induced cells have measurable fluorescence.
OD data do not suggest significant differences in viability from potential toxicity in LB containing 0.5% arabinose, suggesting observed knockdowns are unlikely to result from dead cells with dysfunctional translational machinery for GFP_LVA production.
Conclusions
FACS data of three independent constructs and ODs taken together strongly suggest an observable knockdown of GFP_LVA from arabinose induction. Although these results are very preliminary and require additional verification, they are exciting results that an endogenous, modular method of knockdown at the RNA level is possible, thus potentially enabling elegant and rapid constructs of auto-regulation at the RNA-level that is independent of the biological chassis.
