Difference between revisions of "Part:BBa K4335002"
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==<b>Characterization of Ulink-SZ 2024 team</b>==
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===<b>Introduction</b><br>===
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StayGold is a green fluorescent protein, which has an order of magnitude more photostability than any currently available fluorescent protein. In our project, we tried to analyze whether it could be functional in Escherichia coli BL21(DE3). T7 promoter is a commonly used inducible promoter for protein expression in E. coli BL21(DE3). However, T7 promoter needs to be induced by IPTG, and IPTG is expensive and contains slightly toxic. Thus, we designed a pET-P(BAD)-StayGold plasmid that only needs non-toxic arabinose to induce, and determined whether the pET-P(BAD)-StayGold plasmid could work normally in E. coli BL21(DE3) by detecting the fluorescence expression intensity of fluorescent.
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===<b>Plasmid Construction</b><br>===
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Recombinant plasmid pET29a-StayGold was used as template, and StayGold-For and StayGold-Rev were used as primers to amplify StayGold target gene (654 bp). Using pET-PBAD as template, BAD-vector-For and BAD-vector-Rev as primers, the vector fragment (5037 bp) containing arabinose induced promoter was obtained by amplification. The linearizing vector and the target gene was joined together by one-step cloning. Colony PCR was performed on the transformed colonies using yanzhengFor and yanzhengRev as primers. Positive colonies were transferred and plasmids were extracted.  After sequencing verification, the recombinant plasmid pET-P(BAD)-StayGold was obtained.
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  <img class="bild" src="https://static.igem.wiki/teams/5520/parts/c1.png">
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  <div class="unterschrift"><b>Figure 1 Construction of pET-P(BAD)-StayGold recombinant plasmid</b>
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  <div class="unterschrift"><b>Table 1 Primer sequences</b>
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===<b>Fluorescence intensity detection</b><br>===
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The recombinant plasmid pET-P(BAD)-StayGold was introduced into E. coli BL21(DE3), notated as BAD-StayGold. The transfected Escherichia coli was cultured in LB medium containing 10 mM arabinose till OD600 reached 0.8, 1.0 and 1.2, and the fluorescence intensity was detected. E. coli BL21(DE3) transfected with pET-PBAD empty vector as a control.<br>
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  <img class="bild" src="https://static.igem.wiki/teams/5520/parts/c2.jpg">
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  <div class="unterschrift"><b>Fig.2 Comparison of fluorescence intensity of E. coli BL21(DE3) transfected with recombinant plasmid pET-P(BAD)-StayGold and pET-PBAD empty vector
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BAB-stayGold: E. coli BL21(DE3) harboring pET-P(BAD)-StayGold; Control: E. coli BL21(DE3) harboring pET-PBAD. </b>
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Our results showed that E. coli BL21(DE3) harboring pET-P(BAD)-StayGold achieved efficient expression of stayGold gene with higher fluorescence intensity of 428.52,719.85 and 772.77 under OD600 of 0.8, 1, and 1.2, respectively, compared with the control (251.17, 267.38, 273.85).
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===<b>Exploring the optimal expression temperature of stayGold in E. coli BL21(DE3)</b><br>===
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Further, we measured the change of fluorescence intensity of E. coli BL21(DE3) introduced with pET-P(BAD)-StayGold plasmid by controlling the culture temperature and time. After entering the logarithmic growth phase (OD600~0.5), 10 mM arabinose was added to E. coli BL21(DE3) harboring pET-P(BAD)-StayGold plasmid to induce stayGold gene expression. The fluorescence intensity of stayGold in E. coli BL21(DE3) and the OD600 were determined during the cultivation (Fig 3). (A)-(C) indicates that OD600 has the minimal fluctuation at 30℃. Besides, at 30℃, relative fluorescence intensity maintained the highest than the other two.
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  <img class="bild" src=" https://static.igem.wiki/teams/5520/parts/c4.jpg">
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  <div class="unterschrift"><b>Fig. 3. Relative fluorescence intensity under the growth curve of E. coli BL21(DE3) harboring pET-P(BAD)-StayGold. (A) is incubated at 20℃; (B) is incubated at 30℃. (C) is incubated at 37℃.  </b>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 16:18, 1 October 2024


StayGold, a newly discoverd green fluorescent protein

StayGold, a green fluorescent protein (GFP) , fluoresces in expression. StayGold has an order of magnitude more photostability than any currently available fluorescent protein. Commonly used to label and trace cellular components. BBa_K4335002 is our StayGold gene optimized for the codon of Chlamydomonas reinhardtii.

Additional Materials

StayGold was extracted from Cytaeis uchidae by Atsushi Miyawaki et al. by the RIKEN Center for Brain Science Research on April 25, 2022.

Absorption spectrum and emission spectrum of StayGold.[Image origin]

StayGold and other 15 kinds of fluorescent protein fluorescence intensity changes with time

StayGold has an order of magnitude more light stability than any currently available fluorescent protein and has a cell brightness similar to that of mNeonGreen. Compared with the 15 fluorescent proteins, StayGold showed good photostability.

Usage

We introduced the StayGold gene into the [pTX2040] plasmid. PCrRBCS2 is used as the promoter and TCrRBCS2 is used as the terminator to achieve the expression of the StayGold gene.

Result

Plasmid construction

We designed two primers: Staygold-F and Staygold-R, and confirmed the successful assembly by PCR amplification.

The primers

Primers targeted location

The electrophoretogram of StayGold after amplification

Functional Identification

Fluorescence excitation of positive clones transferred into pTX2040 vector compared with wild-type mCherry, together with DIC field and chlorophyll excitation as fluorescence controls.

Reference

[1] Hirano, M. et al. A highly photostable and bright green fluorescent protein. Nat. Biotechnol. 40, 1132–1142 (2022).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 540
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization of Ulink-SZ 2024 team

Introduction

StayGold is a green fluorescent protein, which has an order of magnitude more photostability than any currently available fluorescent protein. In our project, we tried to analyze whether it could be functional in Escherichia coli BL21(DE3). T7 promoter is a commonly used inducible promoter for protein expression in E. coli BL21(DE3). However, T7 promoter needs to be induced by IPTG, and IPTG is expensive and contains slightly toxic. Thus, we designed a pET-P(BAD)-StayGold plasmid that only needs non-toxic arabinose to induce, and determined whether the pET-P(BAD)-StayGold plasmid could work normally in E. coli BL21(DE3) by detecting the fluorescence expression intensity of fluorescent.

Plasmid Construction

Recombinant plasmid pET29a-StayGold was used as template, and StayGold-For and StayGold-Rev were used as primers to amplify StayGold target gene (654 bp). Using pET-PBAD as template, BAD-vector-For and BAD-vector-Rev as primers, the vector fragment (5037 bp) containing arabinose induced promoter was obtained by amplification. The linearizing vector and the target gene was joined together by one-step cloning. Colony PCR was performed on the transformed colonies using yanzhengFor and yanzhengRev as primers. Positive colonies were transferred and plasmids were extracted. After sequencing verification, the recombinant plasmid pET-P(BAD)-StayGold was obtained.

Figure 1 Construction of pET-P(BAD)-StayGold recombinant plasmid

Table 1 Primer sequences

Fluorescence intensity detection

The recombinant plasmid pET-P(BAD)-StayGold was introduced into E. coli BL21(DE3), notated as BAD-StayGold. The transfected Escherichia coli was cultured in LB medium containing 10 mM arabinose till OD600 reached 0.8, 1.0 and 1.2, and the fluorescence intensity was detected. E. coli BL21(DE3) transfected with pET-PBAD empty vector as a control.

Fig.2 Comparison of fluorescence intensity of E. coli BL21(DE3) transfected with recombinant plasmid pET-P(BAD)-StayGold and pET-PBAD empty vector BAB-stayGold: E. coli BL21(DE3) harboring pET-P(BAD)-StayGold; Control: E. coli BL21(DE3) harboring pET-PBAD. Our results showed that E. coli BL21(DE3) harboring pET-P(BAD)-StayGold achieved efficient expression of stayGold gene with higher fluorescence intensity of 428.52,719.85 and 772.77 under OD600 of 0.8, 1, and 1.2, respectively, compared with the control (251.17, 267.38, 273.85). ===Exploring the optimal expression temperature of stayGold in E. coli BL21(DE3)
=== Further, we measured the change of fluorescence intensity of E. coli BL21(DE3) introduced with pET-P(BAD)-StayGold plasmid by controlling the culture temperature and time. After entering the logarithmic growth phase (OD600~0.5), 10 mM arabinose was added to E. coli BL21(DE3) harboring pET-P(BAD)-StayGold plasmid to induce stayGold gene expression. The fluorescence intensity of stayGold in E. coli BL21(DE3) and the OD600 were determined during the cultivation (Fig 3). (A)-(C) indicates that OD600 has the minimal fluctuation at 30℃. Besides, at 30℃, relative fluorescence intensity maintained the highest than the other two.

Fig. 3. Relative fluorescence intensity under the growth curve of E. coli BL21(DE3) harboring pET-P(BAD)-StayGold. (A) is incubated at 20℃; (B) is incubated at 30℃. (C) is incubated at 37℃.