Difference between revisions of "Part:BBa K1906005"
 
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<partinfo>BBa_K1906005 short</partinfo>
 
<partinfo>BBa_K1906005 short</partinfo>
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This protein is the beta subunit of Qbeta replicase.
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<br>
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<p>This protein is the beta subunit of Qbeta replicase.</p>
  
 
===Characterlization===
 
===Characterlization===
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Whole cell lysate, supernatant and precipitations of cells with different constructs are loaded into SDS-PAGE gels. Figure 3 shows the electrophoresis results of samples that transformed with three different constructs: 1. pETDuet-1-Rep (Expressing β subunit alone) 2. pACYC-TS-TU (co-expressing EF-Ts and EF-Tu) 3. pACYC-TU (Expressing EF-Tu alone) It can be clearly observed that, unpon inducement of IPTG, pETDuet-1-Rep doesn't lead to the abundant production of β subunit. None of the three lanes of cells that transformed with pETDuet-1-Rep display a detectable band at 65.6 kDa, the size of β subunit. The cells that transformed with pACYC-TS-TU and pACYC-TU, however, shows unambiguous production of EF-Ts (position indicated by red arrow) and EF-Tu (position indicated by orange arrow).
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[[File:20161019150339!SDS-TSTU-REP.jpg|200px|thumb|left|alt text]]
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<P>It was quite surprising that plasmids contain this CDS part don't lead to a heavy production of the protein upon the induction. However, this situation can be improved when EF-Ts and EF-Tu are co-expressed with beta subunit.</P>
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[[File:SDS-TSTU-REP.jpg|200px|thumb|left|alt text]]
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<p>Figure 1 is the SDS-PAGE result comparison between cells that were transformed with these three proteins and beta subunit alone.</P>
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[[File:20161019150339!SDS-TSTU-REP.jpg|500px|thumb|left|Figure 1. SDS-PAGE result comparison between three-protein-expressing cells and beta subunit-expressing cells. Wild-type cells were included as a control group. The gel on the left shows the results of cells that had been induced with 2mM IPTG and the gel on the right shows the results of same groups of cells that had not been induced. The blue arrow indicates the band position of beta subunit.]]
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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It can be clearly seen that cells transformed with beta subunit alone had no detectable level of protein expression. However, the expression of beta subunit can be greatly restored when plasmids that carry EF-Tu and EF-Ts are transformed into the cells.  
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<p>However, these results may be further enhanced with the use of a fusion protein design that combines the Qbeta subunit directly in complex with the Ef-Tu and EF-Ts cofactors, as in [[Part:BBa_K3258018]].</p>
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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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When running an SDS-PAGE gel to compare protein expression between the expression of a Qbeta subunit alone versus the expression of the fully linked complex inside E. coli, it can clearly be seen that expression increases significantly with the new part, [[Part:BBa_K3258018]]. 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.
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The general functionality of the Qbeta subunit has also been characterized in vitro.[[File:QBInVitroInitialTest.png|center|400x400px]]
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The functionality and operation of the MDV-Qbeta-Spinach construct ([[Part:BBa_K3258017]]) was tested in Sigma 70 myTXTL Cell Free Cell Lysate. Initial tests were conducted comparing fluorescent output from another construct, mRFP-Spinach, with the fluorescent output of MDV-Qbeta-Spinach. Trials were conducted running reactions with each construct at the same time at 29 degrees Celsius for 18 hours and measuring fluorescent output. Results showed '''significant''' differences in fluorescent output between MDV-Qbeta-Spinach and mRFP-Spinach, suggesting that MDV-Qbeta-Spinach was operational in vitro in the used cell free lysate.
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The importance of MDV regions to replication by the Qbeta subunit for in vitro has also been characterized.
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[[File:QBMDVvsNoMDV.png|center|500x600px]]
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After confirming the functionality of the MDV-Qbeta-Spinach construct with in vitro experimentation, the impact of the MDV regions was tested. Each MDV region ([[BBa_K3258015]] and [[BBa_K3258016]]), or midivariant region, is a RNA sequence experimentally classified in previous literature to be a good substrate for Qbeta binding and replication. Flanking a given gene of interest, especially Qbeta, with the midivariants should make the whole sequence more conducive to replication and amplification by Qbeta. This was tested by comparing the fluorescent output of the MDV-Qbeta-Spinach construct in vitro with trials of a Qbeta-Spinach only construct, which lacked the MDV flanking regions. Run in triplicate, the results suggested a clear improvement of the MDV flanked Qbeta-Spinach over the control Qbeta-Spinach only, with no trial of the MDV-Qbeta-Spinach dipping below the highest performing trial of the Qbeta-Spinach. The differences in amplification of each construct suggested a '''~30% improvement in the amplification rate with the MDV flanked construct''' versus the non-MDV flanked construct.
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Qbeta is also capable of amplifying other genes of interest that are bound by the MDV regions.
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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.
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===Usage and Biology===
 
===Usage and Biology===
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Qbeta replicase is a RNA dependent RNA transcriptase that produces sense strands from antisense strands of RNA. It commonly is found in a complex along 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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Latest revision as of 18:48, 21 October 2019


Beta-subunit of Qbeta replicase holoenzyme

This protein is the beta subunit of Qbeta replicase.

Characterlization

It was quite surprising that plasmids contain this CDS part don't lead to a heavy production of the protein upon the induction. However, this situation can be improved when EF-Ts and EF-Tu are co-expressed with beta subunit.

Figure 1 is the SDS-PAGE result comparison between cells that were transformed with these three proteins and beta subunit alone.

Figure 1. SDS-PAGE result comparison between three-protein-expressing cells and beta subunit-expressing cells. Wild-type cells were included as a control group. The gel on the left shows the results of cells that had been induced with 2mM IPTG and the gel on the right shows the results of same groups of cells that had not been induced. The blue arrow indicates the band position of beta subunit.




















It can be clearly seen that cells transformed with beta subunit alone had no detectable level of protein expression. However, the expression of beta subunit can be greatly restored when plasmids that carry EF-Tu and EF-Ts are transformed into the cells.

However, these results may be further enhanced with the use of a fusion protein design that combines the Qbeta subunit directly in complex with the Ef-Tu and EF-Ts cofactors, as in Part:BBa_K3258018.

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.

When running an SDS-PAGE gel to compare protein expression between the expression of a Qbeta subunit alone versus the expression of the fully linked complex inside E. coli, it can clearly be seen that expression increases significantly with the new part, Part:BBa_K3258018. 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.

The general functionality of the Qbeta subunit has also been characterized in vitro.
QBInVitroInitialTest.png

The functionality and operation of the MDV-Qbeta-Spinach construct (Part:BBa_K3258017) was tested in Sigma 70 myTXTL Cell Free Cell Lysate. Initial tests were conducted comparing fluorescent output from another construct, mRFP-Spinach, with the fluorescent output of MDV-Qbeta-Spinach. Trials were conducted running reactions with each construct at the same time at 29 degrees Celsius for 18 hours and measuring fluorescent output. Results showed significant differences in fluorescent output between MDV-Qbeta-Spinach and mRFP-Spinach, suggesting that MDV-Qbeta-Spinach was operational in vitro in the used cell free lysate.

The importance of MDV regions to replication by the Qbeta subunit for in vitro has also been characterized.

QBMDVvsNoMDV.png

After confirming the functionality of the MDV-Qbeta-Spinach construct with in vitro experimentation, the impact of the MDV regions was tested. Each MDV region (BBa_K3258015 and BBa_K3258016), or midivariant region, is a RNA sequence experimentally classified in previous literature to be a good substrate for Qbeta binding and replication. Flanking a given gene of interest, especially Qbeta, with the midivariants should make the whole sequence more conducive to replication and amplification by Qbeta. This was tested by comparing the fluorescent output of the MDV-Qbeta-Spinach construct in vitro with trials of a Qbeta-Spinach only construct, which lacked the MDV flanking regions. Run in triplicate, the results suggested a clear improvement of the MDV flanked Qbeta-Spinach over the control Qbeta-Spinach only, with no trial of the MDV-Qbeta-Spinach dipping below the highest performing trial of the Qbeta-Spinach. The differences in amplification of each construct suggested a ~30% improvement in the amplification rate with the MDV flanked construct versus the non-MDV flanked construct.

Qbeta is also capable of amplifying other genes of interest that are bound by the MDV regions.

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.

Usage and Biology

Qbeta replicase is a RNA dependent RNA transcriptase that produces sense strands from antisense strands of RNA. It commonly is found in a complex along 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.

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 1317
    Illegal BamHI site found at 1460
    Illegal XhoI site found at 991
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1473
  • 1000
    COMPATIBLE WITH RFC[1000]