Part Collection | Stony-Brook

Introduction

Our part collection consists of three composite parts which are themselves constructed by different combinations of seven new basic parts. It is worth nothing that we also made use of an Anderson promoter (https://parts.igem.org/Part:BBa_J23100), Anderson RBS (https://parts.igem.org/Part:BBa_J61100), and a T1 terminator (https://parts.igem.org/Part:BBa_B0010). The collection consists of the Codon Optimized GST-Ago2 composite part (https://parts.igem.org/Part:BBa_K5090008), the E. coli-based dual regulation system (https://parts.igem.org/Part:BBa_K5090007), and GFP under two lac operators and a kink turn (https://parts.igem.org/Part:BBa_K5090009). We implemented the collection through two plasmids, with the composite Ago2 part in one plasmid and the latter two composite parts in the remaining plasmid.

When the parts are expressed together in E. coli or in an E. coli-based cell free system, the collection is capable of detecting microRNA-326. This is because a microRNA-326 target site is present before the rest of the coding sequence for the dual-regulation construct. Without microRNA-326 present, the dual-regulation system suppresses GFP, as LacI binds to the lacO and L7Ae binds to the kink turn, both in the 5’ UTR of the GFP composite part. When microRNA-326 is present, Ago2, which is expressed constitutively, binds to the miRNA and is guided to the miRNA target site where Ago2 makes a cleavage. This cleavage prevents the expression of the dual-regulation system and thus allows GFP to express. The varied fluorescence of the system provides an indicator of the concentration of miRNA-326.

Part Collection

New Basic Parts in the Collection

Part Name	Type	Description
BBa_K5090000	CDS	Argonaute2, which is codon optimized and has a GST tag to express in E. coli or E. coli-based cell-free systems.
BBa_K5090004	Regulatory	P2A, a "Self cleaving peptide" that allows for the simultaneous expression of multiple proteins from a single RBS and start codon.
BBa_K5090001	CDS	LacI, a transcriptional repressor that binds DNA at the site of lac operators. This specific version was enhanced by Laird et al. to be more effective than wild-type LacI.
BBa_K5090005	Regulatory	P2A, "Self cleaving peptide" that allows for the simultaneous expression of multiple proteins from a single RBS and start codon.
BBa_K5090003	CDS	GFP, a fluorescent protein used as a reporter to indicate gene expression.
BBa_K5090002	CDS	L7Ae, a translational repressor that binds DNA at the site of kink turns.
BBa_K5090006	Regulatory	The reverse-complement of human miRNA-326 (hsa-miR-326), serving as a target site for Argonaute2 when miRNA-326 and the gene to which the target site is attached has been transcribed.

New Composite Parts in the Collection

Part Name	Part Type	Description
BBa_K5090007	Device	Implementation of the "dual regulation system" created by Wang et al., with transcriptional repressor LacI and translational repressor L7Ae for binding with lac operators on DNA and kink turns on mRNA respectively, with the complementary target site for microRNA-326 present within the open reading frame of LacI, and with P2A allowing for LacI and L7Ae to be expressed under the same promoter and RBS while nonetheless being created as separate proteins and for the amino acids coded for by the target site for miRNA-326 to be separated from LacI so as to avoid interference with its function.
BBa_K5090008	Device	Implementation of Codon Optimized GST-Ago2 such that it binds to and cleaves target sites on mRNA strands complementary to the microRNA strands Ago2 loads.
BBa_K5090009	Device	Implementation of GFP under regulation of two lac operators (lacO2) and a kink turn such that its fluorescence is inhibited by LacI and/or L7Ae when present.

As our results demonstrate and our implementation page discusses, an implementation of these parts could be used as part of an accessible and affordable assay that would detect significant markers of B-cell lymphoma sooner than traditional diagnostic methods. This screening would get patients on the path to being tested by more traditional sooner than currently occurs, which is typically not until patients have developed symptoms and lymphoma has become harder to treat.