Difference between revisions of "Part:BBa R0061:Experience"

(→‎iGEM Chiba 2010)
(→‎iGEM Chiba 2010)
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==iGEM Chiba 2010==
 
==iGEM Chiba 2010==
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under construction!
  
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We cheracterized about R0061.We constructed Plux inv-GFP combining R0061 (Plux inv) and E0240 (RBS and GFP).<br>
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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]]. We tested whether the RFP expression is repressed by LuxR and 3OC6HSL.
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Plux inv-GFP and Plac-LuxR was cotransformed by strain of DH10B.The strain was incubeted in LB liquid medium (AHL 0 nM and AHL1000 nM) for 12 h at 37゜C.<br>After incubation,We measured GFP fluorescence(excitation 485 nm,emission 527 nm) and OD600.<br>
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We confirm LuxR repression.
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===Methods===
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*used parts
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**[[Part:BBa_K396012|K396012]] on pSB2K3 (parts which RFP is inserted below R0061)
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**[[Part:BBa_K396011|K396011]] on pSB1A3 (constitutive LuxR generator)
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**GFP regulated by lac promoter on pSB2K3 as a positive control
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*Plux inv-GFP and Plac-LuxR was cotransformed by strain of DH10B.The strain was incubeted in LB liquid medium (AHL 0 nM and AHL1000 nM) for 12 h at 37゜C.<br>After incubation,We measured GFP fluorescence(excitation 485 nm,emission 527 nm) and OD600.<br>
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===Results====
 
[[IMAGE:lux inverter.png|left|thumb|Fig. 1 ]]
 
[[IMAGE:lux inverter.png|left|thumb|Fig. 1 ]]

Revision as of 08:01, 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])


Tokyotech R0061 K395008 graph R0061.jpg

UNIQaddbc2e36d705244-partinfo-00000002-QINU

iGEM Chiba 2010

under construction!
  • 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

  • used parts
    • K396012 on pSB2K3 (parts which RFP is inserted below R0061)
    • K396011 on pSB1A3 (constitutive LuxR generator)
    • GFP regulated by lac promoter on pSB2K3 as a positive control
  • Plux inv-GFP and Plac-LuxR was cotransformed by strain of DH10B.The strain was incubeted in LB liquid medium (AHL 0 nM and AHL1000 nM) for 12 h at 37゜C.
    After incubation,We measured GFP fluorescence(excitation 485 nm,emission 527 nm) and OD600.

Results=

Fig. 1