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

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**Colonies were cultured overnight in LB media with appropriate antibiotics.
 
**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.
 
**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.
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**OD<sub>600</sub>was measured, and 200 µL of the cultures were used to measure GFP fluorescence (excitation 485 nm,emission 527 nm).
+
**OD<sub>600</sub> was measured, and 200 µL of the cultures were used to measure GFP fluorescence (excitation 485 nm,emission 527 nm).
  
 
===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. ]]
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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).
 +
 +
 +
*The Greenberg group [1] (the original paper of this promoter) reported a strong repression of "Plux repression promoter" using '''log phase''' cells.
 +
*The results from iGEM Tokyo Tech 2010 (above in this page) shows about 1/3 repression also using '''log phase''' cells.
 +
*Cox ''et al.'' [2] reported that the "Plux repression promoter"' does not work. This paper measured the cells in a '''stationary phase''' (cultured at 25 ºC for 18 h) as our team did (at 37 ºC for 12 h).
 +
 +
 +
*From these information, we believe that the reason why we couldn't observe the repression is because we performed the experiment using '''stationary phase''' cells.
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 +
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<Br clear="all">
 +
===Reference===
 +
<!-- Replace the PubMed ID's ("pmid=#######") below with the PubMed ID's for your publications.  You can add or remove lines as needed -->
 +
<biblio>
 +
#Egland pmid=10633117
 +
#Cox pmid=18004278
 +
</biblio>
 +
 +
<!--
 +
・このプロモータを報告したGreenberg et alの論文では，蛍光が確認できている。
 +
・東工大も似た実験で，rperessionが確認できている。
 +
・一方で，我々はみられなかった。
 +
・我々と同様に，Cox et.al. も蛍光がみられないと書いている（引用）
 +
 +
これらの実験条件をみると，greenber(the original paper）と東工大は，log phaseで実験をしているのに対し，
 +
Coxと我々は，stationary phaseで実験をしていた。
 +
 +
はっきりと結論づけるには更なる検証が必要だが，
 +
恐らくphaseの違いによって，repressがみれるときとみれないときがあるのだと示唆される。
 +
-->

Revision as of 09:44, 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

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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
    1. K396012 on pSB2K3 (parts which RFP is inserted below R0061)
    2. K396011 on pSB1A3 (constitutive LuxR generator)
    3. J09250 on pSB2K3 (GFP under lac promoter) as a positive control
    4. R0061 on pSB2K3 as a negative control
  • 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.
    • OD600 was measured, and 200 µL of the cultures were used to measure GFP fluorescence (excitation 485 nm,emission 527 nm).

Results & Discussion

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 OD600 and represent the averages of three replicates; error bars, standard deviations.
  • Repression were not observed in the condition we tested (see right Figure).


  • The Greenberg group [1] (the original paper of this promoter) reported a strong repression of "Plux repression promoter" using log phase cells.
  • The results from iGEM Tokyo Tech 2010 (above in this page) shows about 1/3 repression also using log phase cells.
  • Cox et al. [2] reported that the "Plux repression promoter"' does not work. This paper measured the cells in a stationary phase (cultured at 25 ºC for 18 h) as our team did (at 37 ºC for 12 h).


  • From these information, we believe that the reason why we couldn't observe the repression is because we performed the experiment using stationary phase cells.



Reference

<biblio>

  1. Egland pmid=10633117
  2. Cox pmid=18004278

</biblio>