Difference between revisions of "Part:BBa R0061:Experience"
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===User Reviews=== | ===User Reviews=== | ||
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| − | + | <I>Username</I> | |
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| + | Enter the review inofrmation here. | ||
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| + | <I>iGEM Tokyo_Tech 2010</I> | ||
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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. <br><br> | 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. <br><br> | ||
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 OD<sub>590</sub> reached 0.6, added 3OC6HSL. After 3 hours of induction, fluorescence intensity was measured with flow cytometry. <br><br> | 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 OD<sub>590</sub> reached 0.6, added 3OC6HSL. After 3 hours of induction, fluorescence intensity was measured with flow cytometry. <br><br> | ||
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(→[http://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep more information]) | (→[http://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep more information]) | ||
<br>[[IMAGE:Tokyotech R0061 K395008 graph R0061.jpg|300px|none|left|thumb]] | <br>[[IMAGE:Tokyotech R0061 K395008 graph R0061.jpg|300px|none|left|thumb]] | ||
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| − | ==iGEM Chiba 2010== | + | {|width='80%' style='border:1px solid gray' |
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| + | <partinfo>BBa_R0061 AddReview 3</partinfo> | ||
| + | <I>iGEM Chiba 2010</I> | ||
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*To cheracterize R0061 biobrick part, we inserted a GFP reporter ([[Part:BBa_E0240|E0240]]) under R0061 to make [[Part:BBa_K396012|K396012]]. | *To cheracterize R0061 biobrick part, we inserted a GFP reporter ([[Part:BBa_E0240|E0240]]) under R0061 to make [[Part:BBa_K396012|K396012]]. | ||
*We tested whether the GFP expression is repressed by LuxR and 3OC6HSL. | *We tested whether the GFP expression is repressed by LuxR and 3OC6HSL. | ||
| − | + | ====Materials & Methods==== | |
| − | ===Materials & Methods=== | + | |
*plamisd used | *plamisd used | ||
*#[[Part:BBa_K396012|K396012]] on pSB2K3 (parts which GFP is inserted below R0061) | *#[[Part:BBa_K396012|K396012]] on pSB2K3 (parts which GFP is inserted below R0061) | ||
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*#[[Part:BBa_R0061|R0061]] on pSB2K3 as a negative control | *#[[Part:BBa_R0061|R0061]] on pSB2K3 as a negative control | ||
**notes: pSB2K3 is known as a very-low-copy plasmid, only in a lacIq strain. In this experiment we used DH10B which does not have lacIq. | **notes: pSB2K3 is known as a very-low-copy plasmid, only in a lacIq strain. In this experiment we used DH10B which does not have lacIq. | ||
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*strain | *strain | ||
**DH10B | **DH10B | ||
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*procedure | *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) | **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) | ||
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**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. | ||
**OD<sub>600</sub> was measured, and 200 µL of the cultures were used to measure GFP fluorescence with a fluorescence microplate reader(excitation 485 nm,emission 527 nm). | **OD<sub>600</sub> was measured, and 200 µL of the cultures were used to measure GFP fluorescence with a fluorescence microplate reader(excitation 485 nm,emission 527 nm). | ||
| − | + | ====Results==== | |
| − | ===Results | + | [[Image:Chiba R0061.png|right|300px|thumb|'''Figure'''. Fluorescence of GFP under lux repression promoter with/without AHL. p.c. stands for positive control, n.c. stands for negative control. All biobricks are co-transformed with K396011 (constitutive luxR generator). 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| | + | *Repression were not observed in the condition we tested (see right Figure).<br><br> |
| − | *Repression were not observed in the condition we tested (see right Figure). | + | |
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*Egland and Greenberg [1] (the original paper of this promoter) reported a strong repression of "Plux repression promoter" using '''log phase''' cells. | *Egland and Greenberg [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. | *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). | + | *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).<br><br> |
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*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, not log phase cells. | *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, not log phase cells. | ||
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<Br clear="all"> | <Br clear="all"> | ||
| − | + | ====Reference==== | |
| − | ===Reference=== | + | |
<!-- Replace the PubMed ID's ("pmid=#######") below with the PubMed ID's for your publications. You can add or remove lines as needed --> | <!-- Replace the PubMed ID's ("pmid=#######") below with the PubMed ID's for your publications. You can add or remove lines as needed --> | ||
<biblio> | <biblio> | ||
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#Cox pmid=18004278 | #Cox pmid=18004278 | ||
</biblio> | </biblio> | ||
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Revision as of 07:02, 7 November 2010
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UNIQ41354f1850351c66-partinfo-00000000-QINU
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No review score entered. 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. |
|
•••
iGEM Chiba 2010 |
Materials & Methods
Results Figure. Fluorescence of GFP under lux repression promoter with/without AHL. p.c. stands for positive control, n.c. stands for negative control. All biobricks are co-transformed with K396011 (constitutive luxR generator). Bar heights are normalized to OD600 and represent the averages of three replicates; error bars, standard deviations.
Reference<biblio>
</biblio> |
UNIQ41354f1850351c66-partinfo-00000003-QINU