Difference between revisions of "Part:BBa K2842680"

Revision as of 01:32, 18 October 2018

Intein Monomer 1: RFP reporter flanked with orthogonal inteins

This gene encodes a novel split-intein flanked reporter device which enables the use of intein splicing for any protein of interest through SapI digestion. Intein Monomer 1 was created to work in conjunction with its complimentary composite part Intein Monomer 2 to construct a intein polymerisation system.

Usage and Biology

Protein Polymerisation by Split Inteins

Discovered in the late 1980s, inteins are naturally occurring protein segments attached to specific host proteins of unicellular organisms (Protein Engineering Handbook, 2009). Inteins contain both an N- and C-terminal domain, which can be split to allow either half to be bound to unique external proteins. Matching split inteins self-excise from their attached host protein in a trans-splicing reaction (depicted in Figure X), which allows for the ligation of the external proteins through a peptide bond.

Figure 1: Protein trans-splicing

Inteins ligate their flanking sequences with a native peptide bond. These sequences can be either their native exteins or unrelated peptides or proteins. (Thiel et al., Angewandte Communications, 2014)

Intein Monomer 1 utilises a Npu-C and an AceL-TerL-N, which are complementary to the flanked inteins on Intein Monomer 2. The Npu-C split intein is the C-terminal domain fragment from a Nostoc punctiforme (Npu) dnaE gene. It possesses more than 98% trans-splicing efficiency, which is greater splicing activity than other species’ DnaE inteins (Iwai et al., 2006). On the other hand, the AceL-TerL-N, derived from phage genes discovered in antarctic permanently stratified saline lakes, is the smallest N-terminal split intein as it is composed of only 25 amino acids .(Thiel et al., 2014).

Reporter Protein

Intein Monomer 1 encodes the reporter protein, red fluorescent protein (RFP). RFPs derive from various coral species, and are utilised to emit orange and fluorescence under UV-light (Miyawaki et al., 2013). In particular, Intein Monomer 1 uses a highly engineered mutant RFP isolated from Discosoma striata (coral).

Related Entries in the Registry

The 2018 UCL team utilised the previously existing T7 promoter (BBa_J64997), Strep-tag (BBa_K823038), and terminator (BBa_B1006) within the Intein Monomer 1 design. This part works together with Intein Monomer 2 (BBa_K2842690), which was submitted by the 2018 UCL iGEM team.

New Part Status

Other than these specified registered basic parts, the rest of our Intein Monomer 1 construct is made up of newly submitted basic parts by the 2018 UCL iGEM team, therefore potentially making Intein Monomer 1 a novel biobrick. Intein Monomer 1 is also the only Biobrick registered with a AceL-TerL-C intein.

Figure xxx: BsaI digestions

(1) BsaI digested Intein Passenger BBa_K2842669
(2) BsaI digested RFP inteins BBa_K2842680
(3) BsaI digested GFP inteins BBa_K2842690
*edited to show relevant bands

Sequence and Features

Functional Parameters

References

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