Difference between revisions of "Part:BBa K5036019"

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<partinfo>BBa_K5036019 short</partinfo>
 
<partinfo>BBa_K5036019 short</partinfo>
 
==Part Description==
 
==Part Description==
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MS2 is a small viral protein which forms the outer shell of the MS2 bacteriophage. Its ability to bind to specific RNA sequences has made it a valuable tool for studying RNA biology and gene expression and it is frequently used in combination with the MS2 system to purify and analyze RNA-protein complexes this part contain 24 repeats of MS2. while HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication.
  
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it is  a non structural large and multifunctional protein which is encoded by coronavirus. NSP3 plays a crucial role in viral replication through its different activities as protease . Studying NSP3 enables the researchers to gain data about the mechanism of SARS-Cov2 infection and disease progression .
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==Usage=
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we have used 24 repeats of MS2 due to its high activation rate of our switch. MS2 is used to enhances stability through its interaction with MCP, which in turn binds to the MMP9 nanobody. HHR folds spontaneously and cleaves itself to remove the poly A tail, preventing the switch from circularization and thus stopping unintended translation
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==Usage==
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We inserted this part to be attached to the cap of linear RNA.After cutting the poly A tail, the aptamer will be expressed to be attached to MCP. Once the injury occurs ,MMP9 increases and it will mediate the circularization of RNA through its nanobody.
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==literature characterization==
 
==literature characterization==
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To study the effect of a rotavirus protein (RF-NSP3) on cellular translation, researchers chose MA104 cells. These cells are easily infected with rotavirus and better represent what happens during infection. Notably, the RF strain of the virus significantly reduces the translation of a specific type of cellular messenger RNA (poly(A) mRNA) . To create a comparison, they used the established cell line C20b. This line expresses a lower level of the protein of interest (NSP3-RF) compared to MA104 cells infected with the strong RF strain (MOI of 10 for 3 hours, as measured by Western blot) (Figure A). Both C20b and MA104 cells were transfected with reporter messenger RNAs. Then, researchers purified RNA from these cells at two time points: immediately after transfection (time zero) and 6 hours later (time 6). They used a technique called RT-qPCR to quantify the amount of reporter RNA present at each time point (Figure B). Finally, they measured the activity of a protein encoded by the reporter RNA at the 6-hour time point (Figures C to F).
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To understand how various hammerhead  ribozyme motifs affect gene activity, they incorporated eight different motifs into relevant mRNA sequences. To compare results across diverse genetic systems, they assessed reporter gene expression in human cells, baker's yeast (S. cerevisiae), and E. coli bacteria. Well-established plasmid-based gene expression constructs served as their reporter systems.
  
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The study found that even low levels of NSP3 significantly increased the production of proteins from messenger RNAs (mRNAs) with a specific ending sequence (GACC). Interestingly, this boost in protein production from these GACC-ending mRNAs wasn't simply due to NSP3 preserving the mRNA. Additionally, NSP3 itself seemed to promote the translation of both mRNAs with and without a poly(A) tail (poly(A) and nonpoly(A) mRNAs). This suggests that NSP3 might be able to take the place of a cellular complex (PABP-poly(A) complex) normally required to initiate translation.
 
 
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lang=EN style='font-size:11.0pt;line-height:115%'>(Part (A) examined the levels of a viral protein called NSP3 within C20b cells. To achieve this, both C20b and MA104 cells were infected with a specific rotavirus strain (RF) at a set concentration (MOI of 10) for various time points. Western blotting was used to detect both the viral protein (NSP3) and a cellular protein (GAPDH) acting as a reference. The intensity of the signals was measured to compare the amount of NSP3 relative to GAPDH. C20b cells served as the baseline for comparison (ratio set to 1).
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lang=EN style='font-size:11.0pt;line-height:115%'>(A)The researchers placed the HHR motifs at the end of a reporter gene called Renilla luciferase (hRluc) within a vector named psi-CHECK2. The reporter gene is responsible for producing light.
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Parts (B) to (F) focused on reporter mRNAs, which are artificial RNAs used to track cellular processes. These reporter mRNAs (R-, N-, and pA-) were introduced into both MA104 and C20b cells using electroporation. RNA was then extracted from the cells at two time points: immediately after (T0) and 6 hours later (T6). RT-qPCR was employed to quantify the amount of reporter RNA present. Additionally, the activity of a protein produced from the reporter RNA was measured at the 6-hour time point (T6). To account for variations, the activity was normalized to the amount of reporter RNA present at either T0 or T6. Similar to part (A), mock-infected cells with reporter RNA served as the baseline (ratio set to 1).
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(B)They investigated how HHR motifs influence the production of a LacZ gene by inserting them into a specific region (3'-UTR) of a separate Gal4 gene on a plasmid. The LacZ gene, located on a chromosome, is controlled by a promoter that responds to Gal4.
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(C,D) It shows a comparison of how different ribozymes affect gene activity in living cells (in vivo analysis). Black bars represent a reporter gene controlled by a functional HHR motif, while gray bars show the same gene controlled by a non-functional HHR. The control group (Ctrl) lacks any ribozyme sequences. (C) of the figure displays results in human HeLa S3 cells after 18 hours of introducing the genetic material (transfection). (D) shows gene activity in baker's yeast (S. cerevisiae) grown for 18 hours in a special nutrient solution (synthetic complete medium) at room temperature (30°C).
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Revision as of 17:38, 20 September 2024


MS2(x24)-HHR

Part Description

MS2 is a small viral protein which forms the outer shell of the MS2 bacteriophage. Its ability to bind to specific RNA sequences has made it a valuable tool for studying RNA biology and gene expression and it is frequently used in combination with the MS2 system to purify and analyze RNA-protein complexes this part contain 24 repeats of MS2. while HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication.

=Usage

we have used 24 repeats of MS2 due to its high activation rate of our switch. MS2 is used to enhances stability through its interaction with MCP, which in turn binds to the MMP9 nanobody. HHR folds spontaneously and cleaves itself to remove the poly A tail, preventing the switch from circularization and thus stopping unintended translation

this figure illustrates the structure of NSP3 in our switch .

literature characterization

To understand how various hammerhead ribozyme motifs affect gene activity, they incorporated eight different motifs into relevant mRNA sequences. To compare results across diverse genetic systems, they assessed reporter gene expression in human cells, baker's yeast (S. cerevisiae), and E. coli bacteria. Well-established plasmid-based gene expression constructs served as their reporter systems.

(A)The researchers placed the HHR motifs at the end of a reporter gene called Renilla luciferase (hRluc) within a vector named psi-CHECK2. The reporter gene is responsible for producing light. (B)They investigated how HHR motifs influence the production of a LacZ gene by inserting them into a specific region (3'-UTR) of a separate Gal4 gene on a plasmid. The LacZ gene, located on a chromosome, is controlled by a promoter that responds to Gal4. (C,D) It shows a comparison of how different ribozymes affect gene activity in living cells (in vivo analysis). Black bars represent a reporter gene controlled by a functional HHR motif, while gray bars show the same gene controlled by a non-functional HHR. The control group (Ctrl) lacks any ribozyme sequences. (C) of the figure displays results in human HeLa S3 cells after 18 hours of introducing the genetic material (transfection). (D) shows gene activity in baker's yeast (S. cerevisiae) grown for 18 hours in a special nutrient solution (synthetic complete medium) at room temperature (30°C).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 7
    Illegal XhoI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 20
    Illegal AgeI site found at 238
    Illegal AgeI site found at 456
  • 1000
    COMPATIBLE WITH RFC[1000]