Difference between revisions of "Part:BBa K3258018"

 
 
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===Qbeta Replicase Full Fusion Complex===
 
Qbeta replicase beta subunit fused to EF-Tu and EF-Ts, both proteins required for the full replicase to assemble properly. It was shown that this fusion does not require ribosomal protein S1 to be active.
 
Qbeta replicase beta subunit fused to EF-Tu and EF-Ts, both proteins required for the full replicase to assemble properly. It was shown that this fusion does not require ribosomal protein S1 to be active.
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===Background===
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Qbeta replicase is a RNA dependent RNA transcriptase that produces sense strands from antisense strands of RNA. It typically is found in complex with E. coli expression factors EF-Ts and EF-Tu, as well as with the s1 ribosomal subunit. Widely studied because of its properties and its connection to the RNA world hypothesis as an RNA dependent only system capable of self replication, it is also the central protein in the DiCE directed evolution method.
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===Fusion Protein Expression versus Subunit Alone===
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Since the Qbeta holoenzyme is typically found complexed with multiple other proteins and cofactors, whose availability is not guaranteed depending on the environment that Qbeta is used within, there was a potential need identified for a single large linked protein complex featuring Qbeta as well as the EF-Tu and EF-Ts proteins. s1 has been demonstrated as typically being widely available due to its status as a ribosomal subunit as well as not having as significant an impact on Qbeta expression levels with or without its inclusion. In order to compare improvements in this construct for expression in comparison to the previously characterized Qbeta subunit alone, an SDS-PAGE gel was run.
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[[File:Qbgel.png|center|500x500px|SDS-PAGE gel results from comparisons between expression levels within E. coli and in cell free in vitro lysate solution of both the Qbeta subunit alone versus the full linked complex.]]
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Inside E. coli, it can clearly be seen that expression increases significantly with the fusion protein. With cell free lysate the results are inconclusive, potentially a result of the lack of capacity within simple cell free systems for large protein expression, or simply a result of lower activity levels. In either case, more work is needed for in vitro characterization, but the in vivo characterization results demonstrate improved expression.
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===Fusion Protein Replication of Non-Qbeta RNA===
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This fusion Qbeta was also used to amplify GFP flanked with MDV regions in vitro, testing applicability of the system.
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[[File:GFPMDV.png|center|500x500px]]
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As shown in this data, GFP flanked by the MDV regions, in both the Qbeta full linker construct as well as with the subunit alone, were both shown to undergo replication with Qbeta in vitro. This suggests despite the construct's larger size, which could potentially pose a barrier in cell free applications, it is still functional.
  
 
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Latest revision as of 18:58, 21 October 2019


Q-beta EFTu-EFTs fusion protein

Qbeta Replicase Full Fusion Complex

Qbeta replicase beta subunit fused to EF-Tu and EF-Ts, both proteins required for the full replicase to assemble properly. It was shown that this fusion does not require ribosomal protein S1 to be active.

Background

Qbeta replicase is a RNA dependent RNA transcriptase that produces sense strands from antisense strands of RNA. It typically is found in complex with E. coli expression factors EF-Ts and EF-Tu, as well as with the s1 ribosomal subunit. Widely studied because of its properties and its connection to the RNA world hypothesis as an RNA dependent only system capable of self replication, it is also the central protein in the DiCE directed evolution method.

Fusion Protein Expression versus Subunit Alone

Since the Qbeta holoenzyme is typically found complexed with multiple other proteins and cofactors, whose availability is not guaranteed depending on the environment that Qbeta is used within, there was a potential need identified for a single large linked protein complex featuring Qbeta as well as the EF-Tu and EF-Ts proteins. s1 has been demonstrated as typically being widely available due to its status as a ribosomal subunit as well as not having as significant an impact on Qbeta expression levels with or without its inclusion. In order to compare improvements in this construct for expression in comparison to the previously characterized Qbeta subunit alone, an SDS-PAGE gel was run.

SDS-PAGE gel results from comparisons between expression levels within E. coli and in cell free in vitro lysate solution of both the Qbeta subunit alone versus the full linked complex.

Inside E. coli, it can clearly be seen that expression increases significantly with the fusion protein. With cell free lysate the results are inconclusive, potentially a result of the lack of capacity within simple cell free systems for large protein expression, or simply a result of lower activity levels. In either case, more work is needed for in vitro characterization, but the in vivo characterization results demonstrate improved expression.

Fusion Protein Replication of Non-Qbeta RNA

This fusion Qbeta was also used to amplify GFP flanked with MDV regions in vitro, testing applicability of the system.

GFPMDV.png

As shown in this data, GFP flanked by the MDV regions, in both the Qbeta full linker construct as well as with the subunit alone, were both shown to undergo replication with Qbeta in vitro. This suggests despite the construct's larger size, which could potentially pose a barrier in cell free applications, it is still functional.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 535
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 535
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 535
    Illegal BamHI site found at 3399
    Illegal BamHI site found at 3542
    Illegal XhoI site found at 3073
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 535
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 535
    Illegal AgeI site found at 688
    Illegal AgeI site found at 1132
    Illegal AgeI site found at 1537
    Illegal AgeI site found at 3555
  • 1000
    COMPATIBLE WITH RFC[1000]