Composite

Part:BBa_K2842669

Designed by: Catherine Fan, Eloise Nee   Group: iGEM18_UCL   (2018-10-02)


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
Related Device BBa_K1362101
RFC standard RFC10,RFC21,RFC23
& RFC25 compatible
Backbone pSB1C3
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.

Figure 1: Protein trans-splicing
This image depicts the ability of split inteins to form pepide bonds between two proteins of interest while splicing themselves ut of the end product.Inteins ligate their flanking sequences with a native peptide bond. These sequences can be either their native exteins or unrelated peptides or proteins. Figure adapted from Thiel et al., 2014 [1]


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 [1].


Reporter Protein BBa_K2842669 encodes the reporter protein, mScarlet, which is a highly engineered monomeric red fluorescent protein.

Experimental Results

Figure 1: Comparisons of fluorescence/OD600 of BBa_K2842669 and BBa_K1362101

A plate reader was used to measure the fluorescence of whole cell cultures.Plate reader settings: Filters were set to 540/590 , shaking at 200 rpm for 20 h at 25oC. (1-Red) BBa_K2842669 in BL21*(DE3) cells induced with 400 μM IPTG
(2-Orange) BBa_K2842669 in BL21*(DE3) cells without IPTG induction
(3-Grey) BBa_K1362101 expressed in BL21*(DE3) cells />


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 [2][3]. 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.

Figure 2: Expression of IP at varying IPTG concentrations
A plate reader was used to measure the fluorescence of whole cell cultures after induction with various concentrations of ITPG. Plate reader settings: Filters 540/590 , shaking at 200 rpm for 20 h at 25oC. This graph shows the fold change in fluorescence of BBa_K2832669 at a range of IPTG induction concentrations relative to uninduced BBa_K2832669. </p>
Figure 3: SDS-PAGE of BBa_K2832669

We expressed BBa_K2832669 in 50 mL cultures and extracted the intein fusion protein through sonication. The clear lysate was purified on Strep-Tactin columns then SDS-PAGE was performed on the samples. 3A shows the SDS gel when stained with instant blue for 6 hours. 3B shows the fluorescence reading from the gel. (L) Protein standard numbers are in kDa (1) The clear lysate of BBa_K2832669 before purification (2) After purification on Streptactin columns


























Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 941
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal 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

Functional Parameters

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
TGAGTGACAGTTGAAAAGCGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGGTCTCAACGGACGACGCCGGTTACTACATTGA

 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)

101 
201 
301 
MFRTNTNNIKILSPNGFSNFNGIQKVERNLYQHIIFDDDTEIKTSIDHPFGKDKIQARDVKVGDYLNSKKVLYNELVNENIFLYDPINVEKESLYITNGV
VSHNCYNGGRASMVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSF
PEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPV
QMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYKGSSSEWSHPQFEK*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 336 to 337
Strep-tag II: 350 to 357
Amino acid composition:
Ala (A)12 (3.4%)
Arg (R)15 (4.2%)
Asn (N)20 (5.6%)
Asp (D)23 (6.4%)
Cys (C)1 (0.3%)
Gln (Q)11 (3.1%)
Glu (E)29 (8.1%)
Gly (G)33 (9.2%)
His (H)9 (2.5%)
Ile (I)18 (5.0%)
Leu (L)20 (5.6%)
Lys (K)32 (9.0%)
Met (M)12 (3.4%)
Phe (F)18 (5.0%)
Pro (P)17 (4.8%)
Ser (S)22 (6.2%)
Thr (T)21 (5.9%)
Trp (W)4 (1.1%)
Tyr (Y)18 (5.0%)
Val (V)22 (6.2%)
Amino acid counting
Total number:357
Positively charged (Arg+Lys):47 (13.2%)
Negatively charged (Asp+Glu):52 (14.6%)
Aromatic (Phe+His+Try+Tyr):49 (13.7%)
Biochemical parameters
Atomic composition:C1814H2783N487O554S13
Molecular mass [Da]:40694.8
Theoretical pI:6.05
Extinction coefficient at 280 nm [M-1 cm-1]:48820 / 48883 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.73)good (0.68)acceptable (0.56)good (0.65)excellent (0.85)excellent (0.86)
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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References

[1] 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.
[2] 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.
[3] 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

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