Difference between revisions of "Part:BBa K4698008"

Latest revision as of 15:25, 11 October 2023

T7 RNA polymerase has a protein degradation tag attached to its C-terminus

T7 RNA polymerase has a protein degradation tag (degron) attached to its C-terminus, which allows rapid degradation of T7 RNA polymerase, so the T7 promoter cannot be transcribed. Its commonly used in Phage-assisted continuous (PACE).

Usage and Biology

While reviewing CRISPR-related materials, we discovered a particularly interesting evolutionary system- phage-assisted continuous evolution (PACE)[1,2], which can enable continuous directed evolution of gene-coding molecules that can be associated with protein production in E. coli. Then, we found a protein degradation tag-degron[3], that enables rapid hydrolysis of proteins with little functionality, which allows degron to be used in a number of protein co-expression screening systems.

We expressed T7 RNA polymerase (T7 RNAp) in fusion with degron (Fig. 1a), which showed that the T7 RNAp-degron fusion protein was barely able to initiate the transcription of the T7 promoter (Fig. 1b), further demonstrating that degron was able to degrade the T7 RNAp before it could perform its normal function.

Figure 1. a, Schematic representation of the T7 RNAp-degron fusion expression plasmid. b, For the T7 RNAp-degron fusion protein, the efficiency of the T7 promoter, Blank, LB medium. NT, E.coli without the GFP expression plasmid, n=3.

Refrence

1. Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499-503 (2011). 2. Miller, S. M., Wang, T. & Liu, D. R. Phage-assisted continuous and non-continuous evolution. Nature Protocols 15, 4101-4127 (2020). 3. Karzai, A. W., Roche, E. D. & Sauer, R. T. The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue. Nat Struct Biol 7, 449-455 (2000).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <p>While reviewing CRISPR-related materials, we discovered a particularly interesting evolutionary system- phage-assisted continuous evolution (PACE)[1,2], which can enable continuous directed evolution of gene-coding molecules that can be associated with protein production in <i>E. coli</i>. Then, we found a protein degradation tag-degron[3], that enables rapid hydrolysis of proteins with little functionality, which allows degron to be used in a number of protein co-expression screening systems.</p>
-<p>We then expressed T7 RNA polymerase (T7 RNAp) in fusion with degron (Fig. 2a), which showed that the T7 RNAp-degron fusion protein was barely able to initiate the transcription of the T7 promoter, further demonstrating that degron was able to degrade the T7 RNAp before it could perform its normal function.</p>
+<p>We expressed T7 RNA polymerase (T7 RNAp) in fusion with degron (Fig. 1a), which showed that the T7 RNAp-degron fusion protein was barely able to initiate the transcription of the T7 promoter (Fig. 1b), further demonstrating that degron was able to degrade the T7 RNAp before it could perform its normal function.</p>
 <img src="https://static.igem.wiki/teams/4698/wiki/improved-part-t7-rnap.png" class= "center" style="width:500px">
-<p>Figure 2. a, Schematic representation of the T7 RNAp-degron fusion expression plasmid. b, For the T7 RNAp-degron fusion protein, the efficiency of the T7 promoter, Blank, LB medium. NT, <i>E.coli</i> without the GFP expression plasmid, <i>n</i>=3.</p>
+<p>Figure 1. a, Schematic representation of the T7 RNAp-degron fusion expression plasmid. b, For the T7 RNAp-degron fusion protein, the efficiency of the T7 promoter, Blank, LB medium. NT, <i>E.coli</i> without the GFP expression plasmid, <i>n</i>=3.</p>