Difference between revisions of "Part:BBa K5366073"

Revision as of 15:30, 30 September 2024

J23119 promoter-RBS-Bs2-6xHis-T7 termonator

The Bs2 expression plasmid with J23119 as the promoter
(1) Excitation maximum and emission peak
Currently there is limited data on Bs2 in the component library.In order to apply this reporter to practical use, we made contributions to supplement its characteristics, including excitation and emission wavelengths, unit fluorescence intensity in facultative anaerobes. In this study, we used the pET29a plasmid (J23119)(Fig.1)to express the Bs2 protein. In order to expand its application in facultative anaerobic bacteria, we used facultative anaerobic E.coli strain BL21 as expression vector to express Bs2 protein.(Fig.2) After 48 hours of cultivation, the excitation wavelength of Bs2 expressed by pET29a plasmid (J23119) was about 448nm and the emission wavelength was about 509nm(Fig.3).

Fig.1 Construction maps of plasmids of pET29a-Bs2(J23119)

Fig.2 Picture of solid mediumE. coli BL21 with pET29a-Bs2(J23119) on LB solid medium with kanamycin (10μg/ml) and IPTG (0.2 mM), incubated in 37℃ for 24h observed under UV and fluorescence microscopic image taken from the exponential growth phase when OD₆₀₀ was 0.8.

(2)The expression of Bs2 protein in facultative anaerobic bacteria
According to the measured excitation/emission wavelength, we measured the change of unit fluorescence intensity of E. coli BL21 introduced with pET29a-Bs2 plasmid (J23119) by controlling the culture temperature and time. After entering the logarithmic growth phase (OD₆₀₀~0.5), IPTG was added to E. coli with pET29a-Bs2 plasmid (J23119) to induce Bs2 gene expression. OD₆₀₀ and fluorescence intensity were measured every 1h, and OD₆₀₀ and fluorescence intensity were measured every 2h after entering the stable phase.The unit fluorescence intensity of Bs2 in E. coli BL21 was determined (Fig.4).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 287
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 520
Illegal PstI site found at 287
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 287
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 287
1000
COMPATIBLE WITH RFC[1000]

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-   <div class="unterschrift"><b>Fig.2 Picture of solid medium</b><i>E. coli</i> BL21 with pET29a-Bs2(J23119) on LB solid medium with kanamycin (10μg/ml) and IPTG (0.2 mM), incubated in 37℃ for 24h observed under UV and fluorescence microscopic image taken from the exponential growth phase when OD600 was 0.8.
+   <div class="unterschrift"><b>Fig.2 Picture of solid medium</b><i>E. coli</i> BL21 with pET29a-Bs2(J23119) on LB solid medium with kanamycin (10μg/ml) and IPTG (0.2 mM), incubated in 37℃ for 24h observed under UV and fluorescence microscopic image taken from the exponential growth phase when OD<sub>600</sub> was 0.8.
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 <b>(2)The expression of Bs2 protein in facultative anaerobic bacteria</b><br>
-According to the measured excitation/emission wavelength, we measured the change of unit fluorescence intensity of <i>E. coli</i> BL21 introduced with pET29a-Bs2 plasmid (J23119) by controlling the culture temperature and time. After entering the logarithmic growth phase (OD600~0.5), IPTG was added to <i>E. coli</i> with pET29a-Bs2 plasmid (J23119) to induce Bs2 gene expression. OD600 and fluorescence intensity were measured every 1h, and OD600 and fluorescence intensity were measured every 2h after entering the stable phase.The unit fluorescence intensity of Bs2 in E. coli BL21 was determined (Fig.4).
+According to the measured excitation/emission wavelength, we measured the change of unit fluorescence intensity of <i>E. coli</i> BL21 introduced with pET29a-Bs2 plasmid (J23119) by controlling the culture temperature and time. After entering the logarithmic growth phase (OD<sub>600</sub>~0.5), IPTG was added to <i>E. coli</i> with pET29a-Bs2 plasmid (J23119) to induce Bs2 gene expression. OD<sub>600</sub> and fluorescence intensity were measured every 1h, and OD<sub>600</sub> and fluorescence intensity were measured every 2h after entering the stable phase.The unit fluorescence intensity of Bs2 in <i>E. coli</i> BL21 was determined (Fig.4).
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                   <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/30-119.png"><br>
                   <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/37-119.png"><br>
-                  <b>Fig.4. RFU and RFU/OD600 under the growth curve of <i>E. coli</i> with pET29a-Bs2 plasmid (J23119)</b>(A) is incubated at 20℃; (B) is incubated at 30℃. (C) is incubated at 37℃.(A)-(C) indicate that OD600 has the minimal fluctuation at 30℃, however at 30℃ RFU maintained the highest than the other two.
+                  <b>Fig.4. RFU and RFU/OD600 under the growth curve of <i>E. coli</i> with pET29a-Bs2 plasmid (J23119)</b>(A) is incubated at 20℃; (B) is incubated at 30℃. (C) is incubated at 37℃.(A)-(C) indicate that OD<sub>600</sub> has the minimal fluctuation at 30℃, however at 30℃ RFU maintained the highest than the other two.
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