Difference between revisions of "Part:BBa R0061:Experience"
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==iGEM Chiba 2010== | ==iGEM Chiba 2010== | ||
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*To cheracterize R0061 biobrick part, we inserted an RFP reporter ([[Part:BBa_E0240|E0240]]) below R0061 to make [[Part:BBa_K396012|K396012]]. | *To cheracterize R0061 biobrick part, we inserted an RFP reporter ([[Part:BBa_E0240|E0240]]) below R0061 to make [[Part:BBa_K396012|K396012]]. | ||
*We tested whether the RFP expression is repressed by LuxR and 3OC6HSL. | *We tested whether the RFP expression is repressed by LuxR and 3OC6HSL. | ||
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===Results & Discussion=== | ===Results & Discussion=== | ||
[[Image:Chiba R0061.png|right|400px|thumb|'''Figure'''. Fluorescence of lux repressor - GFP with/without AHL. p.c. stands for positive control, n.c. stands for negative control. Bar heights are normalized to OD<sub>600</sub> and represent the averages of three replicates; error bars, standard deviations. ]] | [[Image:Chiba R0061.png|right|400px|thumb|'''Figure'''. Fluorescence of lux repressor - GFP with/without AHL. p.c. stands for positive control, n.c. stands for negative control. Bar heights are normalized to OD<sub>600</sub> and represent the averages of three replicates; error bars, standard deviations. ]] | ||
| + | under construction! | ||
*Repression were not observed in the condition we tested (see right Figure). | *Repression were not observed in the condition we tested (see right Figure). | ||
Revision as of 08:55, 6 November 2010
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Applications of BBa_R0061
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iGEM Tokyo_Tech 2010
iGEM Tokyo_Tech 2010
In order to characterize R0061, Plux repression promoter, we constructed K395101 combining R0061 and K121013, which is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1 and used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.
Overnight cultures of reporter strains grown at 37 °C containing appropriated antibiotics were diluted at least 1:100 and incubated at 37 °C as fresh cultures. After their OD590 reached 0.6, added 3OC6HSL. After 3 hours of induction, fluorescence intensity was measured with flow cytometry.
After 3 hours of induction by 3OC6HSL, the expression of GFP with 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL.
We confirmed that this promoter works correctly.
(→[http://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep more information])
UNIQ0dfe92ce1c66371a-partinfo-00000002-QINU
iGEM Chiba 2010
- To cheracterize R0061 biobrick part, we inserted an RFP reporter (E0240) below R0061 to make K396012.
- We tested whether the RFP expression is repressed by LuxR and 3OC6HSL.
Methods
- plamisd used
- strain
- DH10B
- procedure
- plasmid 1 and 2 was cotransformed into DH10B. (plasmid 2 and 3 was used as a positive control, 3 and 4 as a negative control)
- Colonies were cultured overnight in LB media with appropriate antibiotics.
- Overnight cultures were diluted to 1:100 and incubated in a fresh LB medium (containing 0 or 1 uM 3OC6HSL and antibiotics) at 37 ºC for 12 h.
- OD600was measured, and 200 µL of the cultures were used to measure GFP fluorescence (excitation 485 nm,emission 527 nm).
Results & Discussion
under construction!
- Repression were not observed in the condition we tested (see right Figure).