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>
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<b>(1) Excitation maximum and emission peak</b><br>
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<b>Introduction</b><br>
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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.
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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>
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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).
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<b>Plasmid Construction</b><br>
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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.
 
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  <div class="unterschrift"><b>Table 1 Primer sequences</b>
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<b>Excitation maximum and emission peak of Bs2 in <i>E.coli</i> BL21(DE3)</b><br>
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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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   <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.
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   <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>
 
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  <div class="unterschrift"><b>Fig 2.A fluorescence microscopic image taken from the exponential growth phase when OD<sub>600</sub>=0.8.  </b>
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                 <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.
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                 <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.
 
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<b>(2)The expression of Bs2 protein in facultative anaerobic bacteria</b><br>
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<b>Fluorescence intensity detection</b><br>
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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).
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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>  
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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>
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  <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>
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<b>The optimization of cultivation temperature on Bs2 expression in <i>E. coli BL21</i>(DE3)</b><br>
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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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                 <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.
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                 <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.
 
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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.


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.
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]