Difference between revisions of "Part:BBa K649104:Experience"

Latest revision as of 15:18, 28 October 2011

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Applications of BBa_K649104

We characterized BBa_K649104 and BBa_K117002 in lsrR(-) strain of E.coli.

Fluorescence intensity of BBa_K649104 was much higher than that of promoterless-gfp(negative control).
Strain used in this assay lacks lsrR.
This work is done by Takuya Tsubaki.

Fluorescence intensity of our lsrA promoter-gfp(BBa_K649104) was much higher than that of promoterless-gfp(negative control), showing that our new lsrA promoter(BBa_K649100) works. In spite of no LsR repression, gene transcription does not take place sufficiently. On the other hand, fluorescence intensity of lsrA promoter-gfp((BBa_K117002)-(BBa_J54103)) was almost the same as promoterless-gfp(negative control), showing that lsrA promoter(BBa_K117002) does not work properly. Gene transcription takes place sufficiently. The difference between lsrA promoter(BBa_K117002) and lsrA promoter(BBa_K649100) is whether promoter contains CRP binding site or not. Our lsrA promoter(BBa_K649100) contains this site but lsrA promoter(BBa_K117002) does not. According to Wang(2005) et al, cAMP-CRP directly binds to the upstream of promoter and stimulates expression of the lsr operon.

…TGTGAtctattcgTCGGA…

CRP recognition sites are shown in capital letter.BBa_K649100 contains this sites.

The working part we created allow the use of AI-2 as a signaling molecule, which is a very powerful tool to build complex Synthetic Biology systems.

[sample]

Ptet-gfp on pSB1A2(JD22597)

Promoterless-gfp on pSB6A1(JD22597)

PlsrA-gfp on pSB1A2(BBa_K649104)(JD22597)

PlsrA-gfp on pSB1A2(BBa_K117002-gfp)(JD22597)

JD22597 is a strain lacking lsrR.

[Method]

1.Overnight cultures of reporter strains grown at 37°C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37°C as fresh cultures.

2. After their OD590 reached 0.15, the fresh cultures were diluted 1:100.

3. After 4-hour incubation at 37°C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer.

After four hours from OD590 reaching 0.15, we measured OD.
This work is done by Takuya Tsubaki.

User Reviews

UNIQ9326e2eb93685524-partinfo-00000000-QINU UNIQ9326e2eb93685524-partinfo-00000001-QINU

@@ Line 4: / Line 4: @@
 ===Applications of BBa_K649104===
-[[Image:K649104_OD1.png|thumb|center|700px]]
+We characterized BBa_K649104 and BBa_K117002 in <i>lsrR</i>(-) strain of <i>E.coli</i>.
-[[Image:PlsrA_activity.png|thumb|center|700px|Median fluorescence intensity(MFI) of BBa_K649104 was much higher than that of promoterless-gfp(negative control).<br>Amp　FSC：E01　SSC:350　FL1:750<br>This work is done by Takuya Tsubaki.]]
+[[Image:PlsrA2.png|thumb|center|600px|Fluorescence intensity of BBa_K649104 was much higher than that of promoterless-gfp(negative control).<br>Strain used in this assay lacks <i>lsrR</i>.<br>This work is done by Takuya Tsubaki.]]
-Fluorescence intensity  of our promoter lsrA-gfp(BBa_649104) was much higher than that of  promoterless-gfp(negative control), showing that our new promoter lsrA(BBa_649100) works. On the other hand, fluorescence intensity of PlsrA(BBa_117002)-gfp was almost the same as a promoterless-gfp(negative control), showing that BBa_K117002 does not work properly.The difference between promoter lsrA(BBa_K117002) and promoter lsrA(BBa_649100) is whether promoter contains CRP binding site or not. Our promoter lsrA(BBa_649100) contains this site. According to Wang(2005) et al, cAMP-CRP directly binds to the upstream of promoter and stimulates expression of the lsr operon.
+Fluorescence intensity  of our lsrA promoter-<i>gfp</i>(BBa_K649104) was much higher than that of  promoterless-<i>gfp</i>(negative control), showing that our new lsrA promoter(BBa_K649100) works. In spite of no LsR repression, gene transcription does not take place sufficiently. On the other hand, fluorescence intensity of lsrA promoter-<i>gfp</i>((BBa_K117002)-(BBa_J54103)) was almost the same as promoterless-<i>gfp</i>(negative control), showing that lsrA promoter(BBa_K117002) does not work properly. Gene transcription takes place sufficiently. The difference between lsrA promoter(BBa_K117002) and lsrA promoter(BBa_K649100) is whether promoter contains CRP binding site or not. Our lsrA promoter(BBa_K649100) contains this site but lsrA promoter(BBa_K117002) does not. According to Wang(2005) et al, cAMP-CRP directly binds to the upstream of promoter and stimulates expression of the lsr operon.
@@ Line 15: / Line 15: @@
 CRP recognition sites are shown in capital letter.BBa_K649100 contains this sites.
+The working part we created allow the use of AI-2 as a signaling molecule, which is a very powerful tool to build complex Synthetic Biology systems.
 [sample]
-pSB1A2 Ptet-gfp(JD22597)
+Ptet-<i>gfp</i> on pSB1A2(JD22597)
-pSB6A1 promoterless-gfp(JD22597)
+Promoterless-<i>gfp</i> on pSB6A1(JD22597)
-pSB1A2 PlsrA-gfp(BBa_K649104)(JD22597)
+PlsrA-<i>gfp</i> on pSB1A2(BBa_K649104)(JD22597)
-pSB1A2 PlsrA-gfp(BBa_K11702-GFP)(JD22597)
+PlsrA-<i>gfp</i> on pSB1A2(BBa_K117002-<i>gfp</i>)(JD22597)
+JD22597 is a strain lacking <i>lsrR</i>.
 [Method]
-.Overnight cultures of reporter strains grown at 37 °C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37 °C as fresh cultures.
+.Overnight cultures of reporter strains grown at 37&deg;C in LB medium containing appropriate antibiotics were diluted 1:100 into 3 ml of LB medium and were incubated at 37&deg;C as fresh cultures.
 . After their OD590 reached 0.15, the fresh cultures were diluted 1:100.
-. After 4-hour incubation at 37 °C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer.
+. After 4-hour incubation at 37&deg;C, 1 ml of each culture was moved to 1.6ml tube and its fluorescence intensity was measured with a flow cytometer.
+[[Image:Our_new_lsrA_promoter_activity%28BBa_K649104%29.png|thumb|center|400px|After four hours from OD590 reaching 0.15, we measured OD.<br>This work is done by Takuya Tsubaki.]]
 ===User Reviews===