Difference between revisions of "Part:BBa K5366073"

Latest revision as of 14:06, 1 October 2024

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

The Bs2 expression plasmid with J23119 as the promoter
Introduction
To optimize the expression of Bs2, we harnessed 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 without any addition of inducers, such as IPTG. 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). We assessed its characteristics, including excitation and emission wavelengths, unit fluorescence intensity in facultative anaerobes, and imaging of facultative anaerobes harboring recombinant plasmids under UV light.
Plasmid Construction
The recombinant plasmid pET29a-BS2 (including T7 promoter) was used as template, and j23119-For-20240719 and j23119-Rev-20240719 were used as primers to linearize vector (5695 bp) and one-step assembly was performed directly to construct PET29a-BS2 (J23119) (Fig 1). Using yanzheng23119-For and yanzheng23119-Rev as primers, colony PCR (734 bp) was performed on the transformed colonies. Positive colonies were transferred and plasmid was extracted. After sequencing verification, the recombinant plasmid PET29a-BS2 (J23119) was obtained.

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

Table 1 Primer sequences

Excitation maximum and emission peak of Bs2 in E.coli BL21(DE3)
Further, we employed the facultative anaerobic E. coli strain BL21(DE3) to express Bs2 in plasmid pET29a-Bs2 (J23119). The pictures of recombiant strains on the LB plate and fluorescence microscopic image indicated the successful expression of Bs2 (Fig 2). Following 48 hours of cultivation, the excitation wavelength of Bs2 expressed from the pET29a-Bs2 (J23119) recombinant plasmid was approximately 448 nm, with an emission wavelength around 509 nm, as measured by a multifunctional microplate detector (Fig 4).

Fig 2.E.coli BL21 with pET29a-Bs2 (J23119) on LB solid medium with kanamycin(10μg/ml) incubated in 37 ℃ for 24h. All was observed under UV.

Fig 2.A fluorescence microscopic image taken from the exponential growth phase when OD₆₀₀=0.8.

Fluorescence intensity detection
The recombinant plasmid pET29-Bs2 (J23119) was introduced into Escherichia coli strain BL21(DE3) (notated as J23119). The transfected Escherichia coli was cultured in LB medium till OD₆₀₀ reached 0.8, 1.0 and 1.2, and the fluorescence intensity was detected. Escherichia coli BL21(DE3) transfected with pET29a empty vector as a blank control (notated as BL21).
Compared with the blank control (261.97, 287.54, 304.34), J23119 achieved efficient expression of Bs2 gene with high RFU of 566.44, 1943.8 and 2767.61 under OD₆₀₀ of 0.8, 1 and 1.2 (Fig 5).

Fig.5 Comparison of RFU of Escherichia coli strain BL21(DE3) transfected with recombinant plasmid pET29-Bs2(J23119) and that transfected with pET29a empty vector (RFU: Relative fluorescence unit)

The optimization of cultivation temperature on Bs2 expression in E. coli BL21(DE3)
Finally, we adjusted cultivation temperature and time to evaluate changes in unit fluorescence intensity of E. coli BL21(DE3) with the pET29a-Bs2 (J23119) plasmid. OD₆₀₀ and fluorescence intensity were recorded hourly until steady state, after which measurements were taken every two hours. The unit fluorescence intensity of Bs2 in E. coli BL21 with recombinant plasmids pET29a-Bs2 (J23119) was determined (Fig. 4). Fig 4A-4C indicated that the recombiant strain showed better fluorescence intensity at 30℃ and 37℃.

Fig.6. 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]

@@ Line 4: / Line 4: @@
 The Bs2 expression plasmid with J23119 as the promoter<br>
-<b>(1) Excitation maximum and emission peak</b><br>
+<b>Introduction</b><br>
-To enhance the relative fluorescence unit intensity, we replaced the T7 promoter with the J23119 promoter.
+To optimize the expression of Bs2, we harnessed 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 <i>E. coli</i> without any addition of inducers, such as IPTG. 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). We assessed its characteristics, including excitation and emission wavelengths, unit fluorescence intensity in facultative anaerobes, and imaging of facultative anaerobes harboring recombinant plasmids under UV light.<br>
-J23119 is a prokaryotic constitutive super strong promoter expression vector plasmid, which offers several advantages:
+<b>Plasmid Construction</b><br>
-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>
+The recombinant plasmid pET29a-BS2 (including T7 promoter) was used as template, and j23119-For-20240719 and j23119-Rev-20240719 were used as primers to linearize vector (5695 bp) and one-step assembly was performed directly to construct PET29a-BS2 (J23119) (Fig 1). Using yanzheng23119-For and yanzheng23119-Rev as primers, colony PCR (734 bp) was performed on the transformed colonies. Positive colonies were transferred and plasmid was extracted. After sequencing verification, the recombinant plasmid PET29a-BS2 (J23119) was obtained.
-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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+<html>
+<style>
+    .bild {max-width: 60% ; height: auto;}
+</style>
+<p>
+  <img class="bild" src="https://static.igem.wiki/teams/5366/part/table-1.png">
+  <div class="unterschrift"><b>Table 1 Primer sequences</b>
+  </div>
+</p>
+</html>
+<b>Excitation maximum and emission peak of Bs2 in <i>E.coli</i> BL21(DE3)</b><br>
+Further, we employed the facultative anaerobic <i>E. coli</i> strain BL21(DE3) to express Bs2 in plasmid pET29a-Bs2 (J23119). The pictures of recombiant strains on the LB plate and fluorescence microscopic image indicated the successful expression of Bs2 (Fig 2). Following 48 hours of cultivation, the excitation wavelength of Bs2 expressed from the pET29a-Bs2 (J23119) recombinant plasmid was approximately 448 nm, with an emission wavelength around 509 nm, as measured by a multifunctional microplate detector (Fig 4).<br>
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    <img class="bild" src="https://static.igem.wiki/teams/5366/part/c119.png">
-   <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.
+   <div class="unterschrift"><b>Fig 2.<i>E.coli</i> BL21 with pET29a-Bs2 (J23119) on LB solid medium with kanamycin(10μg/ml) incubated in 37 ℃ for 24h. All was observed under UV. </b>
    </div>
 </p>
+</html>
+<html>
+<style>
+    .bild {max-width: 60% ; height: auto;}
+</style>
+<p>
+  <img class="bild" src="https://static.igem.wiki/teams/5366/part/2.png">
+  <div class="unterschrift"><b>Fig 2.A fluorescence microscopic image taken from the exponential growth phase when OD<sub>600</sub>=0.8.   </b>
+  </div>
+</p>
+</html>
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                   <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/ex119.png">
                   <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.4 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>
 </center>
 </html>
-<b>(2)The expression of Bs2 protein in facultative anaerobic bacteria</b><br>
+<b>Fluorescence intensity detection</b><br>
-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).
+The recombinant plasmid pET29-Bs2 (J23119) was introduced into <i>Escherichia coli</i> strain BL21(DE3) (notated as J23119). The transfected <i>Escherichia coli</i> was cultured in LB medium till OD<sub>600</sub> reached 0.8, 1.0 and 1.2, and the fluorescence intensity was detected. <i>Escherichia coli</i> BL21(DE3) transfected with pET29a empty vector as a blank control (notated as BL21).<br>
+Compared with the blank control (261.97, 287.54, 304.34), J23119 achieved efficient expression of Bs2 gene with high RFU of 566.44, 1943.8 and 2767.61 under OD<sub>600</sub> of 0.8, 1 and 1.2 (Fig 5). <br>
+<html>
+<style>
+    .bild {max-width: 60% ; height: auto;}
+</style>
+<p>
+  <img class="bild" src="https://static.igem.wiki/teams/5366/part/10-2.png">
+  <div class="unterschrift"><b>Fig.5 Comparison of RFU of <i>Escherichia coli</i> strain BL21(DE3) transfected with recombinant plasmid pET29-Bs2(J23119) and that transfected with pET29a empty vector (RFU: Relative fluorescence unit)</b>
+  </div>
+</p>
+</html>
+<b>The optimization of cultivation temperature on Bs2 expression in <i>E. coli BL21</i>(DE3)</b><br>
+Finally, we adjusted cultivation temperature and time to evaluate changes in unit fluorescence intensity of <i>E. coli</i> BL21(DE3) with the pET29a-Bs2 (J23119) plasmid. OD<sub>600</sub> and fluorescence intensity were recorded hourly until steady state, after which measurements were taken every two hours. The unit fluorescence intensity of Bs2 in <i>E. coli</i> BL21 with recombinant plasmids pET29a-Bs2 (J23119) was determined (Fig. 4). Fig 4A-4C indicated that the recombiant strain showed better fluorescence intensity at 30℃ and 37℃.<br>
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                   <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/c30119.png"><br>
                   <img style="width:65%;" src="https://static.igem.wiki/teams/5366/part/c37119.png"><br>
-                  <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.
+                  <b>Fig.6. 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>
 </center>