Difference between revisions of "Part:BBa K2842669"

Latest revision as of 02:50, 18 October 2018

mScarlet reporter with TerL-C intein on the N terminus

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

Functional Parameters

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