Difference between revisions of "Part:BBa K5366073"
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The Bs2 expression plasmid with J23119 as the promoter<br>
 
The Bs2 expression plasmid with J23119 as the promoter<br>
 
<b>(1) Excitation maximum and emission peak</b><br>
 
<b>(1) Excitation maximum and emission peak</b><br>
−
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.
+
To enhance the relative fluorescence unit intensity, we replaced the T7 promoter with the J23119 promoter.
−
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 <i>E.coli</i> 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).
+
J23119 is a prokaryotic constitutive super strong promoter expression vector plasmid, which offers several advantages:
 +
Firstly, the J23119 promoter is one of the strongest constitutive promoters reported in its wild-type form, capable of efficiently driving the expression of foreign genes in E. coli. Secondly, the addition of an UP element (designated as UPa) upstream of the core J23119 promoter further enhances expression efficiency, resulting in a 1.34-fold increase in relative fluorescence units (RFU).<br>
 +
Currently, there is limited data available on Bs2 within the component library. To facilitate the practical application of this reporter, we aimed to enhance its characteristics, including excitation and emission wavelengths as well as unit fluorescence intensity in facultative anaerobes.<br>
 +
In this study, we utilized the pET29a plasmid (J23119) (Fig. 1) to express the Bs2 protein. To broaden its application in facultative anaerobic bacteria, we employed the facultative anaerobe <i>E.coli</i> strain BL21 as the expression vector for the Bs2 protein (Fig. 2). After 48 hours of cultivation, the excitation wavelength of the Bs2 protein expressed by the pET29a plasmid (J23119) was found to be approximately 448 nm, while the emission wavelength measured around 509 nm (Fig. 3).
 
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<center>
 
<center>
 
<figure>
 
<figure>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/exbs2.png">
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/ex119.png">
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/embs2.png"><br>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/em119.png"><br>
 
                 <b>Fig.3.Excitation maximum and emission peak (RFU: Relative fluorescence unit).</b>The Ex Wavelength in nm (Em: 520) indicates that there is one peak values of excite wavelength and it is 448 nm. The Em Wavelength in nm (Ex: 448 nm) shows excluding the impact of three peaks value of excite wavelength, the emission wavelength is around 509 nm.
 
                 <b>Fig.3.Excitation maximum and emission peak (RFU: Relative fluorescence unit).</b>The Ex Wavelength in nm (Em: 520) indicates that there is one peak values of excite wavelength and it is 448 nm. The Em Wavelength in nm (Ex: 448 nm) shows excluding the impact of three peaks value of excite wavelength, the emission wavelength is around 509 nm.
 
</figure>
 
</figure>
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</html>
 
</html>
 
<b>(2)The expression of Bs2 protein in facultative anaerobic bacteria</b><br>
 
<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 (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).
+
Based on the measured excitation and emission wavelengths, we assessed the changes in unit fluorescence intensity of E. coli BL21 transformed with the pET29a-Bs2 plasmid (J23119) by controlling the culture temperature and duration. Once the culture reached the logarithmic growth phase (OD<sub>600</sub> ~ 0.5), IPTG was added to induce the expression of the Bs2 gene. Both OD<sub>600</sub> and fluorescence intensity were measured every hour during this phase. After entering the stable phase, these measurements were taken every two hours. The unit fluorescence intensity of Bs2 in <i>E. coli</i> BL21 was subsequently determined (Fig. 4).
 
<html>
 
<html>
 
<center>
 
<center>
 
<figure>
 
<figure>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/20-119.png"><br>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/c20119.png"><br>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/30-119.png"><br>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/c30119.png"><br>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/37-119.png"><br>
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                 <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/c37119.png"><br>
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                 <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.
+
                 <b>Fig.4. RFU and RFU/OD<sub>600</sub> under the growth curve of <i>E. coli</i> with pET29a-Bs2 plasmid (J23119).</b>The first is incubated at 20℃; The second is incubated at 30℃. The third is incubated at 37℃.Under incubation at 30℃ Celsius,RFU reached their maximum values.Under incubation at 30℃ Celsius,OD<sub>600</sub> reached their maximum values.
 
</figure>
 
</figure>
 
</center>
 
</center>

Revision as of 08:45, 1 October 2024


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

The Bs2 expression plasmid with J23119 as the promoter
(1) Excitation maximum and emission peak
To enhance the relative fluorescence unit intensity, we replaced the T7 promoter with the J23119 promoter. J23119 is a prokaryotic constitutive super strong promoter expression vector plasmid, which offers several advantages: Firstly, the J23119 promoter is one of the strongest constitutive promoters reported in its wild-type form, capable of efficiently driving the expression of foreign genes in E. coli. Secondly, the addition of an UP element (designated as UPa) upstream of the core J23119 promoter further enhances expression efficiency, resulting in a 1.34-fold increase in relative fluorescence units (RFU).
Currently, there is limited data available on Bs2 within the component library. To facilitate the practical application of this reporter, we aimed to enhance its characteristics, including excitation and emission wavelengths as well as unit fluorescence intensity in facultative anaerobes.
In this study, we utilized the pET29a plasmid (J23119) (Fig. 1) to express the Bs2 protein. To broaden its application in facultative anaerobic bacteria, we employed the facultative anaerobe E.coli strain BL21 as the expression vector for the Bs2 protein (Fig. 2). After 48 hours of cultivation, the excitation wavelength of the Bs2 protein expressed by the pET29a plasmid (J23119) was found to be approximately 448 nm, while the emission wavelength measured around 509 nm (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.


Fig.3.Excitation maximum and emission peak (RFU: Relative fluorescence unit).The Ex Wavelength in nm (Em: 520) indicates that there is one peak values of excite wavelength and it is 448 nm. The Em Wavelength in nm (Ex: 448 nm) shows excluding the impact of three peaks value of excite wavelength, the emission wavelength is around 509 nm.
(2)The expression of Bs2 protein in facultative anaerobic bacteria
Based on the measured excitation and emission wavelengths, we assessed the changes in unit fluorescence intensity of E. coli BL21 transformed with the pET29a-Bs2 plasmid (J23119) by controlling the culture temperature and duration. Once the culture reached the logarithmic growth phase (OD600 ~ 0.5), IPTG was added to induce the expression of the Bs2 gene. Both OD600 and fluorescence intensity were measured every hour during this phase. After entering the stable phase, these measurements were taken every two hours. The unit fluorescence intensity of Bs2 in E. coli BL21 was subsequently determined (Fig. 4).



Fig.4. RFU and RFU/OD600 under the growth curve of E. coli with pET29a-Bs2 plasmid (J23119).The first is incubated at 20℃; The second is incubated at 30℃. The third is incubated at 37℃.Under incubation at 30℃ Celsius,RFU reached their maximum values.Under incubation at 30℃ Celsius,OD600 reached their maximum values.

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]