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Team:British Columbia/Jammer
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| - | + | =Jammer: Endogenous, Modular RNA Knockdown= | |
| + | |||
| + | 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 | ||
| + | grown. 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 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 presense 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 | ||
| + | measureable fluorescence. | ||
| + | |||
| + | OD data do not suggest significant differences in viability from potential | ||
| + | toxicity in LB containing 0.5% arabinose, implying 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. | ||
Revision as of 06:48, 20 October 2009
Contents |
Jammer: Endogenous, Modular RNA Knockdown
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 grown. 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 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 presense 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
measureable fluorescence.
OD data do not suggest significant differences in viability from potential toxicity in LB containing 0.5% arabinose, implying 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.
