Difference between revisions of "Part:BBa K5398002"
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<partinfo>BBa_K5398002 short</partinfo>
 
<partinfo>BBa_K5398002 short</partinfo>
  
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<p>This part is a component of the <i>td</i> intron (5' side), an intron of the <i>td</i> gene from T4 phage belonging to group IE, which can form a circular mRNA (cmRNA) to make the ribosomes repeatedly translate the extron. This strategy can be used for expression of long peptides containing tandem repeats, like fibrous proteins.</p>
+
<p>This part is a component of the <i>td</i> intron (5' side), an intron of the <i>td</i> gene from T4 phage belonging to group I introns, which can form a circular mRNA (cmRNA) to make the ribosomes repeatedly translate the extron. This year, we utilized the <i>td</i> intron to produce the squid ring proteins with various long tandem repeats.</p>
 +
<p>We expored different production and purification strategies of target protein produced by cmRNA and examined the function of protein. Simultaneously, we also test the optimal nucleotide length of the <i>td</i> exon between the 5' and 3' intron.</p>
  
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<p> <b>The mechanism of cmRNA </b> </p>
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___TOC___
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<P>Due to the special internal structure, the <i>td</i> intron can circularize the exon to form a back-splice junction (BSJ) in a reaction catalyzed by guanosine. To ensure that the ribosomes do not translate the ORF of gene of interest (GOI) from unprocessed linear mRNA, the ribosome binding sequence (RBS) and start codon ATG were placed downstream of GOI coding sequence. Consequently, the regulatory sequences were located upstream of the coding sequence only after circularization of the mRNA. To purify the resulting polypeptides, a His tag was incorporated into the GOI. If the mRNA is circularized, the ribosome could circle the cmRNA, producing a long repeating polypeptide (Fig. 1). </p>
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===Introduction===
 +
<P>Due to special internal structure, the <i>td</i> intron, also called RNA cyclase ribozyme, can splice themselves out without assistance from the spliceosome or other proteins, and instead rely on a free guanosine nucleotide to initiate the splicing reaction <i>in vivo</i>. This process results in joining of the flanking exons and circularization of the intervening intron to produce an intronic circRNA (Fig. 1). So it is a strategy to produce circular RNAs <i>in vivo</i>.
  
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<html>
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<html lang="zh">
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<center><img src="https://static.igem.wiki/teams/5398/cmrna/design-of-a-circular-mrna.webp"with="600" height="" width="450" height=""/></center>
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<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 */
 +
            padding: 20px; /* 内边距 */
 +
            margin: 20px auto; /* 外边距，自动居中 */
 +
            width: 800px; /* 模块宽度 */
 +
            text-align: center; /* 内容居中 */
 +
            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
    </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/cmrna/intron-mechanism.webp" width="700" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 1 Mechanism of group I introns. (GOMES R M O da S et al. 2024) </b></p>
 +
    </div>
 +
</body>
 
</html>
 
</html>
  
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<p style="text-align: center!important;"><b>Fig. 1 Design of a circular mRNA based on <i>td</i> flanking introns.
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<p>Therefore, an engineering circular mRNA (cmRNA) was designed by employing the RNA cyclase ribozyme mechanism. This elaborate design of cmRNA sequence circularizes the exon to form a back-splice junction (BSJ) in a reaction catalyzed by guanosine. To ensure that the ribosomes do not translate the ORF of gene of interest (GOI) from unprocessed linear mRNA, the ribosome binding sequence (RBS) and start codon ATG were placed downstream of GOI coding sequence. Consequently, the regulatory sequences were located upstream of the coding sequence only after circularization of the mRNA. To purify the resulting polypeptides, a His tag was incorporated into the GOI. If the mRNA is circularized, the ribosome could circle the cmRNA, producing a long repeating polypeptide (Fig. 2). </p>
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</b></p>
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<p>In our project, we used this part to design a cmRNA to produce the squid ring teeth proteins with five tandem repeats (TRn5).</p>
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<html lang="zh">
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<head>
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<p>The constructed plasmid pET29a(+)-cmRNA was transformed into <i>E.coli</i> BL21 (DE3) and recombinant proteins were expressed using LB medium (Fig. 2). </p>
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    <meta charset="UTF-8">
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    <meta name="viewport" content="width=device-width, initial-scale=1.0">
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<html>
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    <style>
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<center><img src="https://static.igem.wiki/teams/5398/cmrna/the-plasmid-map-of-pet29a-cmrna-1.webp"with="500" height="" width="375" height=""/></center>
+
        .module {
 +
            border: 1px solid #ccc; /* 边框 /
 +
*            padding: 20px; /* 内边距 /
 +
*            margin: 20px auto; /* 外边距，自动居中 /
 +
*            width: 800px; /* 模块宽度 /
 +
*            text-align: center; /* 内容居中 /
 +
*            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
    </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/trn5/sequence-of-trn.webp" width="700" height="auto" alt="Protein purification">
 +
        <p><b>Fig. 2 Design of a circular mRNA based on <i>td</i> flanking introns.</b></p>
 +
    </div>
 +
</body>
 
</html>
 
</html>
  
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<p style="text-align: center!important;"><b>Fig. 2 The plasmid map of pET29a(+)-cmRNA.
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===Usage and Biology===
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</b></p>
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<p>In our project, given the positive correlation between number of repeat units and magnitude of cohesive force, we designed a circular mRNA on which the OFR of TRn5 (BBa_K5398001) between the 3' and 5' intron of <i>td</i> gene from T4 phage (BBa_K5398002 and BBa_K5398003). This strategy could use short sequences to express highly repetitive squid ring teeth proteins. A self-cleaving RNA cyclase ribozyme was incorporated to form the circular mRNAs, allowing ribosomes to repeatedly translate the sequence of interest and producing proteins with different repeat numbers, thus we could obtain proteins with exceptional self-healing properties.</p>
  
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<p>After incubation at 37℃ for 5h and 16℃ for 20h, respectively, we found that the TRn expression level in both supernatant and pellet was pretty low. The TRn polypeptide was composed of repeating units with a size of 16 kDa, which was formed by the ribosome traveling one round along the circular mRNA. Due to uncertainty of the round number that the ribosome traveled, TRn sample was a mixture of proteins with various sizes that formed a ladder on the gel. According to the protein marker, we supposed that the sizes of the proteins ranged from about 8 to 96 kDa, indicating that the ribosome could travel along the cmRNA at least 6 rounds (Fig. 3).</p>
+
===Characterization===
 +
====Protein expression====
 +
<p>The synthetic plasmid pET29a(+)-cmRNA(TRn5) was transformed into <i>E.coli</i> BL21 (DE3) and recombinant proteins were expressed using LB medium (Fig. 3). </p>
  
−
<html>
+
<html lang="zh">
−
<center><img src="https://static.igem.wiki/teams/5398/cmrna/sds-page-1.webp"with="500" height="" width="375" height=""/></center>
+
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 +
    <style>
 +
        .module {
 +
            border: 1px solid #ccc; /* 边框 /
 +
*            padding: 20px; /* 内边距 /
 +
*            margin: 20px auto; /* 外边距，自动居中 /
 +
*            width: 800px; /* 模块宽度 /
 +
*            text-align: center; /* 内容居中 /
 +
*            box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */
 +
        }
 +
    </style>
 +
</head>
 +
<body>
 +
    <div class="module">
 +
        <img src="https://static.igem.wiki/teams/5398/cmrna/map-of-pet29a-cmrna-trn5.webp" width="700" height="auto" alt="Protein purification">
 +
          <p><b>Fig. 3 The plasmid map of pET29a(+)-cmRNA(TRn5).</b></p>
 +
    </div>
 +
</body>
 
</html>
 
</html>
  
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<p style="text-align: center!important;"><b>Fig. 3 SDS-PAGE of expression products of cmRNA. Lane 1: marker; lanes 2 to 5: whole-cell lysate, supernatant, pellet and diluted pellet from induced cells at 37℃, respectively; Lane 6: marker; lanes 7 to 9: whole-cell lysate, supernatant and pellet from induced cells at 16℃, respectively.
 
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</b></p>
 
  
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<p>Then, we purified TRn on a HiTrap Ni-NTA column. However, the TRn expression level was too low to verify by SDS-PAGE (Fig. 4).</p>
 
  
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<html>
 
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<center><img src="https://static.igem.wiki/teams/5398/cmrna/sds-page-2.webp"with="600" height="" width="450" height=""/></center>
 
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</html>
 
  
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<p style="text-align: center!important;"><b>Fig. 4 SDS-PAGE of expression products of cmRNA purified by IMAC. Lanes 1 to 6, induced cell sample at 16℃; lane 1: sample after being bound to Ni-NTA resin; lane 2: sample eluted with 20 mM Tris-HCl; lane 3 to 6: sample eluted with 50, 150,300 and 500 mM imidazole; lane 7: marker; Lanes 8 to 13, induced cell sample at 37℃; lane 8: sample after being bound to Ni-NTA resin; lane 9: sample eluted with 20 mM Tris-HCl; lane 10 to 13: sample eluted with 50, 150 and 300 mM imidazole.
 
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</b></p>
 
  
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<p>To optimize the TRn expression, we reviewed plenty of literature, from which we found that TRn could easily be dissolved in 5% acetic acid (pH≈3) due to the existence of Histidine. Thus, we used a new protocol to obtain the purified TRn. Solubilized in 5% acetic acid, the bands of TRn were still seen as a form of ladder, which is a symbol of multi-circle translation of cmRNA. (Fig. 5)</p>
 
  
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<html>
 
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<center><img src="https://static.igem.wiki/teams/5398/cmrna/sds-page-3.webp"with="400" height="" width="300" height=""/></center>
 
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</html>
 
  
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<p style="text-align: center!important;"><b>Fig. 5 SDS-PAGE of expression products of cmRNA. Lane 1: marker; lanes 2 to 4: whole-cell lysate, supernatant and pellet from induced cells at 37℃, respectively; lane 5: sample washed with 5% acetic acid.
 
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</b></p>
 
  
 +
 +
====Self-healing test====
  
  
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==== Reference ====
 
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<p>[1] LIU L, WANG P, ZHAO D, et al. Engineering Circularized mRNAs for the Production of Spider Silk Proteins[J]. <i>Appl. Environ. Microbiol.</i>, 2022, 88(8): e00028-22.</p>
 
−
<p>[2] PERRIMAN R, ARES M. Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo[J]. <i>RNA</i>, 1998, 4(9): 1047-1054.</p>
 
−
<p>[3] LEE S O, XIE Q, FRIED S D. Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins[J]. <i>ACS Cent. Sci.</i>, 2021, 7(10): 1736-1750.</p>
 
−
<p>[4] OBI P, CHEN Y G. The design and synthesis of circular RNAs[J]. <i>Methods</i>, 2021, 196: 85-103.</p>
 
−
<p>[5] GOMES R M O da S, SILVA K J G da, THEODORO R C. Group I introns: Structure, splicing and their applications in medical mycology[J]. <i>Genet. Mol. Biol.</i>, 2024, 47: e20230228.</p>
 
  
  
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<partinfo>BBa_K5398002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5398002 SequenceAndFeatures</partinfo>
  
 +
==== Reference ====
 +
<p>[1] LIU L, WANG P, ZHAO D, et al. Engineering Circularized mRNAs for the Production of Spider Silk Proteins[J]. <i>Appl. Environ. Microbiol.</i>, 2022, 88(8): e00028-22.</p>
 +
<p>[2] PERRIMAN R, ARES M. Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo[J]. <i>RNA</i>, 1998, 4(9): 1047-1054.</p>
 +
<p>[3] LEE S O, XIE Q, FRIED S D. Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins[J]. <i>ACS Cent. Sci.</i>, 2021, 7(10): 1736-1750.</p>
 +
<p>[4] OBI P, CHEN Y G. The design and synthesis of circular RNAs[J]. <i>Methods</i>, 2021, 196: 85-103.</p>
 +
<p>[5] GOMES R M O da S, SILVA K J G da, THEODORO R C. Group I introns: Structure, splicing and their applications in medical mycology[J]. <i>Genet. Mol. Biol.</i>, 2024, 47: e20230228.</p>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 16:46, 20 September 2024


The 5' intron of td gene from T4 phage

This part is a component of the td intron (5' side), an intron of the td gene from T4 phage belonging to group I introns, which can form a circular mRNA (cmRNA) to make the ribosomes repeatedly translate the extron. This year, we utilized the td intron to produce the squid ring proteins with various long tandem repeats.

We expored different production and purification strategies of target protein produced by cmRNA and examined the function of protein. Simultaneously, we also test the optimal nucleotide length of the td exon between the 5' and 3' intron.

_ _

Introduction

Due to special internal structure, the td intron, also called RNA cyclase ribozyme, can splice themselves out without assistance from the spliceosome or other proteins, and instead rely on a free guanosine nucleotide to initiate the splicing reaction in vivo. This process results in joining of the flanking exons and circularization of the intervening intron to produce an intronic circRNA (Fig. 1). So it is a strategy to produce circular RNAs in vivo.

Protein purification

Fig. 1 Mechanism of group I introns. (GOMES R M O da S et al. 2024)

<p>Therefore, an engineering circular mRNA (cmRNA) was designed by employing the RNA cyclase ribozyme mechanism. This elaborate design of cmRNA sequence circularizes the exon to form a back-splice junction (BSJ) in a reaction catalyzed by guanosine. To ensure that the ribosomes do not translate the ORF of gene of interest (GOI) from unprocessed linear mRNA, the ribosome binding sequence (RBS) and start codon ATG were placed downstream of GOI coding sequence. Consequently, the regulatory sequences were located upstream of the coding sequence only after circularization of the mRNA. To purify the resulting polypeptides, a His tag was incorporated into the GOI. If the mRNA is circularized, the ribosome could circle the cmRNA, producing a long repeating polypeptide (Fig. 2).

Protein purification

Fig. 2 Design of a circular mRNA based on td flanking introns.

Usage and Biology

In our project, given the positive correlation between number of repeat units and magnitude of cohesive force, we designed a circular mRNA on which the OFR of TRn5 (BBa_K5398001) between the 3' and 5' intron of td gene from T4 phage (BBa_K5398002 and BBa_K5398003). This strategy could use short sequences to express highly repetitive squid ring teeth proteins. A self-cleaving RNA cyclase ribozyme was incorporated to form the circular mRNAs, allowing ribosomes to repeatedly translate the sequence of interest and producing proteins with different repeat numbers, thus we could obtain proteins with exceptional self-healing properties.

Characterization

Protein expression

The synthetic plasmid pET29a(+)-cmRNA(TRn5) was transformed into E.coli BL21 (DE3) and recombinant proteins were expressed using LB medium (Fig. 3).

Protein purification

Fig. 3 The plasmid map of pET29a(+)-cmRNA(TRn5).





Self-healing test

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Unknown

Reference

[1] LIU L, WANG P, ZHAO D, et al. Engineering Circularized mRNAs for the Production of Spider Silk Proteins[J]. Appl. Environ. Microbiol., 2022, 88(8): e00028-22.

[2] PERRIMAN R, ARES M. Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo[J]. RNA, 1998, 4(9): 1047-1054.

[3] LEE S O, XIE Q, FRIED S D. Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins[J]. ACS Cent. Sci., 2021, 7(10): 1736-1750.

[4] OBI P, CHEN Y G. The design and synthesis of circular RNAs[J]. Methods, 2021, 196: 85-103.

[5] GOMES R M O da S, SILVA K J G da, THEODORO R C. Group I introns: Structure, splicing and their applications in medical mycology[J]. Genet. Mol. Biol., 2024, 47: e20230228.