mScarlet reporter with TerL-C intein on the N terminus
|Intein Passenger: mScarlet with TerL-C intein|
|Function||Functionalise and cap intein polymers|
|Use in||E. coli cells|
|Chassis Tested||DH5α cells|
|Abstraction Hierarchy||Composite Device|
|RFC standard||RFC10,RFC21,RFC23 |
& RFC25 compatible
|Submitted by||UCL iGEM 2018|
This DNA construct encodes a C-terminal segment of the AceL-TerL intein fused to the N-terminus of a mScarlet reporter protein which contains a C-terminal StrepTag for purification. The AceL-TerL intein acts as a protein ligase that forms a peptide bond between mScarlet and another protein bound to a complementary split intein. During this process the intein splices itself out of the protein and is not present in the final fusion protein. This construct is a modular platform for the creation of split intein fusion proteins through the SapI restriction sites located immediately upstream and downstream of the mScarlet reporter. BBa_K2832680 was designed to be used in conjunction with this construct as it contains the corresponding N-terminal intein segment to enable intein trans-splicing.
Usage and Biology
Discovered in the late 1980s, inteins arewere found to be naturally occurring protein segments attached to specific host proteins of unicellular organisms (Protein Engineering Handbook, 2009). Split-inteins are expressed in two separate proteins as N or C terminal domains. Either half can be bound to unique external proteins to create intein fusion proteins. Matching split inteins self-excise from their attached host protein in a trans-splicing reaction (depicted in Figure 1), which allows for the ligation of the external proteins through a peptide bond.
BBa_K2842669 utilises an AceL-TerL-C split intein, which are complementary to one of the the split inteins on BBa_K2842680. AceL-TerL-C is derived from phage genes discovered in antarctic permanently stratified saline lakes. It’s corresponding N terminal intein is the smallest N-terminal split intein as it is composed of only 25 amino acids .
Reporter Protein BBa_K2842669 encodes the reporter protein, mScarlet, which is a highly engineered monomeric red fluorescent protein.
This construct (BBa_K2842669) is also an improvement of the part BBa_K1362101. Both parts were designed to allow for the modular design and assembly of intein fusion proteins, but our part has distinct advantages and improvements. We used mScarlet as our reporter instead of mRFP1 as it is known to be brighter than mRFP1 . Our reporter protein mScarlet has an 11-fold increase in fluorescence/OD600 compared to BBa_K1362101 when driven by the expression from the T7 promoter (Fig3). Our construct also has a dual function as both an assembly construct and reporter for building new inteins, and as an intein fusion by itself. Due to this, it can be used both to test intein functionality in other constructs and to build new ones.
We measured the expression of BBa_K2842669 with a BMG Fluostar plate reader by analysis of its fluorescence at varying IPTG concentrations. We found that mScarlet is very well expressed when produced from an IPTG-inducible T7 promoter. Increasing IPTG concentrations from 400 μM to 800 μM had little effect on expression levels and cell growth rate (Fig1A, Fig1B), indicating that IPTG concentrations at 400 μM are sufficient to overproduce our protein. We also tested the modularity of the construct by replacing mScarlet with with two new sequences to create the biobricks BBa_K2842710 and BBa_K2842720. Diagnostic digests with BsaI ((Figure 2) and the sequencing results confirmed that the cloning was successful.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NotI site found at 941
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI site found at 1224
Illegal BsaI.rc site found at 28
Illegal SapI.rc site found at 419
Illegal SapI site found at 1130
|Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0|
|Nucleotide sequence in RFC 10: (underlined part encodes the protein)|
GCTTCTACAAACGCGGCTTCTTCCAAAGAGACCTAATACGACTCACTATAGGGGTTGTGAGCGGATAACAACCGCAAATTTAAGGATTCTCAAAAATGTTCCGC ... TTCGAAAAA
ORF from nucleotide position 96 to 1166 (excluding stop-codon)
|Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)|
|Sequence features: (with their position in the amino acid sequence, see the list of supported features)|
|Amino acid composition:|
|Amino acid counting|
|Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges|
|Alignments (obtained from PredictProtein.org)|
There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
|Predictions (obtained from PredictProtein.org)|
|There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.|
| The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.|
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 Thiel IV, Volkmann G, Pietrokovski S and Mootz HD. (2014) An atypical naturally split intein engineered for highly efficient protein labeling. Angewandte Communications, Int. Ed., 53: 1306-1310.
 Bindels DS, Haarbosch L, van Weeren L, Postma M, Wieser KE, Mastop M, et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods. 2017;14(1):53-6.
 Robert E. Campbell, Oded Tour, Amy E. Palmer, Paul A. Steinbach, Geoffrey S. Baird, David A. Zacharias, Roger Y. Tsien, A monomeric red fluorescent protein Proceedings of the National Academy of Sciences Jun 2002, 99 (12) 7877-7882