Difference between revisions of "Part:BBa K5082005"
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RNA is a common type of biological molecule. In cells, RNAs exist in many forms including messenger RNA (mRNA), ribosomal RNA (rRNA), translational RNA (tRNA), etc. Although RNA molecules are single-stranded, unlike DNA, they still form secondary structures through intramolecular complementary base-pairing to minimize free-energy (−ΔG/nt) and become more thermodynamically stable. mRNA secondary structures are dependent on factors including base sequence, protein binding, and guanine-cytosine content. mRNA secondary structures with −ΔG/nt≥0.3 are defined to be highly structured while those with −ΔG/nt ≤0.2 are defined to be poorly structured [1]. mRNA secondary structures are often found in non-coding regions, especially in the 3’UTR [2]. Highly structured 3’UTR are abbreviated as HSU while poorly structured 3’ UTR are abbreviated as PSU. Although HSU regions are non-coding, they serve vital roles in the regulation of gene expression [1]. Previously, the EIF3B gene has been reported to contain an HSU structure [3]. Therefore, this sequence can be used to regulate gene expression once fused downstream to a gene. The secondary structure of an RNA sequence can be predicted by computer software such as mFold or ViennaRNA [4]. The predicted secondary structure for EIF3B-HSU is shown in Figure 1. | RNA is a common type of biological molecule. In cells, RNAs exist in many forms including messenger RNA (mRNA), ribosomal RNA (rRNA), translational RNA (tRNA), etc. Although RNA molecules are single-stranded, unlike DNA, they still form secondary structures through intramolecular complementary base-pairing to minimize free-energy (−ΔG/nt) and become more thermodynamically stable. mRNA secondary structures are dependent on factors including base sequence, protein binding, and guanine-cytosine content. mRNA secondary structures with −ΔG/nt≥0.3 are defined to be highly structured while those with −ΔG/nt ≤0.2 are defined to be poorly structured [1]. mRNA secondary structures are often found in non-coding regions, especially in the 3’UTR [2]. Highly structured 3’UTR are abbreviated as HSU while poorly structured 3’ UTR are abbreviated as PSU. Although HSU regions are non-coding, they serve vital roles in the regulation of gene expression [1]. Previously, the EIF3B gene has been reported to contain an HSU structure [3]. Therefore, this sequence can be used to regulate gene expression once fused downstream to a gene. The secondary structure of an RNA sequence can be predicted by computer software such as mFold or ViennaRNA [4]. The predicted secondary structure for EIF3B-HSU is shown in Figure 1. | ||
| − | + | https://static.igem.wiki/teams/5407/eif3b-hsu.png | |
| − | + | Figure 1. EIF3B-HSU secondary structure predicted by RNAfold [5]. | |
| − | + | ||
Design | Design | ||
In our project, we found that the G3BP1 protein was overexpressed in gastric cancer (GC) cells [6]. Meanwhile, G3BP1 could bind with HSU structures and lead to mRNA degradation [7]. Therefore, we fused the EIF3B-HSU sequence downstream to reporter genes: GFP and luciferase, to monitor G3BP1 levels and hence diagnose GC. The experimental outline is shown in Figure 2. | In our project, we found that the G3BP1 protein was overexpressed in gastric cancer (GC) cells [6]. Meanwhile, G3BP1 could bind with HSU structures and lead to mRNA degradation [7]. Therefore, we fused the EIF3B-HSU sequence downstream to reporter genes: GFP and luciferase, to monitor G3BP1 levels and hence diagnose GC. The experimental outline is shown in Figure 2. | ||
| − | + | https://static.igem.wiki/teams/5407/eif3b-hsu-2.png | |
| − | Figure 2. Experimental outline. (A) GFP sensor system. (B) Luciferase sensor system. | + | Figure 2.Experimental outline. (A) GFP sensor system. (B) Luciferase sensor system. |
Revision as of 09:37, 31 August 2024
RNA is a common type of biological molecule. In cells, RNAs exist in many forms including messenger RNA (mRNA), ribosomal RNA (rRNA), translational RNA (tRNA), etc. Although RNA molecules are single-stranded, unlike DNA, they still form secondary structures through intramolecular complementary base-pairing to minimize free-energy (−ΔG/nt) and become more thermodynamically stable. mRNA secondary structures are dependent on factors including base sequence, protein binding, and guanine-cytosine content. mRNA secondary structures with −ΔG/nt≥0.3 are defined to be highly structured while those with −ΔG/nt ≤0.2 are defined to be poorly structured [1]. mRNA secondary structures are often found in non-coding regions, especially in the 3’UTR [2]. Highly structured 3’UTR are abbreviated as HSU while poorly structured 3’ UTR are abbreviated as PSU. Although HSU regions are non-coding, they serve vital roles in the regulation of gene expression [1]. Previously, the EIF3B gene has been reported to contain an HSU structure [3]. Therefore, this sequence can be used to regulate gene expression once fused downstream to a gene. The secondary structure of an RNA sequence can be predicted by computer software such as mFold or ViennaRNA [4]. The predicted secondary structure for EIF3B-HSU is shown in Figure 1.
Figure 1. EIF3B-HSU secondary structure predicted by RNAfold [5].
Design In our project, we found that the G3BP1 protein was overexpressed in gastric cancer (GC) cells [6]. Meanwhile, G3BP1 could bind with HSU structures and lead to mRNA degradation [7]. Therefore, we fused the EIF3B-HSU sequence downstream to reporter genes: GFP and luciferase, to monitor G3BP1 levels and hence diagnose GC. The experimental outline is shown in Figure 2.
Figure 2.Experimental outline. (A) GFP sensor system. (B) Luciferase sensor system.