Overview

Our composite parts, consists of three variants of reporter systems and an antisense oligonucleotide (ASO) system. In particular, our ASO system (BBa_K5401006) is a strong contender for the best composite part due to its innovative design and practical applications. The ASO system provides precise control over gene regulation by inhibiting translation, which is essential for fine-tuning gene expression in various experimental contexts. The utilisation of an ASO helps to regulate the expression of T7RNAP, thereby allowing for the stable expression of the polymerase. In addition, our three reporter variants demonstrate the versatile monitoring of gene expression, enabling researchers to choose the most effective system for their specific needs.

Furthermore, our basic parts entail the two intricate components of our ASO system, the antisense oligonucleotide targeted against the +4 to +27 of the wild-type T7RNAP mRNA sequence, as well as the Hfq scaffold sequence to recruit the Hfq protein for promoting the binding between sRNA and its complementary mRNA target. Three ancestral sequences derived from ancestral reconstruction were also added as part due to their potential as possible distant homologs to the wild-type T7RNAP.

Taken together, our parts continue to contribute to the current inventory of biological systems.

Composite Part

Composite Part	Plasmid Code	Short Description	Long Description	Source	Design Consideration	Subparts
BBa_K5401000	C1	pUC_T7Promoter_eCFP	High-copy number plasmid encoding for enhanced cyan fluorescent protein (eCFP) under T7 Promoter. The plasmid was utilised as part of a reporter system to evaluate the efficiency of T7 RNA polymerase and its variants.	Molecular cloning using ligation; sequence of individual parts were derived from iGEM Registry.	The design of the plasmid contains a high-copy number plasmid backbone (BBa_J428353), T7 Promoter (BBa_K3457003), RBS (BBa_J428032), eCFP (BBa_E0020), and T7 Terminator (BBa_J428091).	BBa_J428353 - pJUMP28-1A (sfGFP) | BBa_K3457003 - T7 Promoter | BBa_J428032 - Ribosomal Binding Site (RBS) | BBa_E0020 - eCFP | BBa_J428091 - T7Te
BBa_K5401002	C2	pBR322_T7Promoter_eCFP	Medium-copy number plasmid encoding for enhanced cyan fluorescent protein (eCFP) under T7 Promoter. The plasmid was utilised as part of a reporter system to evaluate the efficiency of T7 RNA polymerase and its variants.	Molecular cloning using ligation; sequence of individual parts were derived from iGEM Registry.	The design of the plasmid contains a medium-copy number plasmid backbone (BBa_J428341), T7 Promoter (BBa_K3457003), RBS (BBa_J428032), eCFP (BBa_E0020), and T7 Terminator (BBa_J428091).	BBa_J428341 - pBR322 Backbone | BBa_K3457003 - T7 Promoter | BBa_J428032 - Ribosomal Binding Site (RBS) | BBa_E0020 - eCFP | BBa_J428091 - T7Te
BBa_K5401003	C3	p15A_T7Promoter_eCFP	Low-copy number plasmid encoding for enhanced cyan fluorescent protein (eCFP) under T7 Promoter. The plasmid was utilised as part of a reporter system to evaluate the efficiency of T7 RNA polymerase and its variants.	Molecular cloning using ligation; sequence of individual parts were derived from iGEM Registry.	The design of the plasmid contains a low-copy number plasmid backbone (BBa_J428341), T7 Promoter (BBa_K3457003), RBS (BBa_J428032), eCFP (BBa_E0020), and T7 Terminator (BBa_J428091).	BBa_J428341 - p15A Backbone | BBa_K3457003 - T7 Promoter | BBa_J428032 - Ribosomal Binding Site (RBS) | BBa_E0020 - eCFP | BBa_J428091 - T7Te
BBa_K5401006	ASO System	Antisense Oligonucleotide System (ASO)	Antisense Oligonucleotide (ASO) system is designed to bind to specific mRNA sequences, effectively suppressing the translation of target proteins. By silencing mRNA, ASOs reduce the expression of genes.	Ordered Gblocks based on the designed sequences, cloned into plasmid.	The design consists of the sequences from the targeted gene of expression in a plasmid with wild-type T7 RNA polymerase.	BBa_K5401004 | Hfq scaffold sequence | BBa_K5401005 | T7 RNA polymerase (T7RNAP) antisense oligonucleotide (ASO)

Basic Part

Basic Part	Part Name	Usage and Biology	Characterization
BBa_K5401004	Hfq scaffold sequence	The Hfq scaffold sequence recruits the Hfq protein, an RNA-binding protein that functions primarily as a riboregulator. The Hfq protein facilitates the binding between sRNA and complementary mRNA sequence, thereby inhibiting translation and regulating expression.	The combination of our antisense oligonucleotide (ASO), the lac promoter, and the T7-expressing plasmid (plasmid 1c) resulted in more stable T7RNAP expression and a lower frequency of insertions compared to the previous plasmid (plasmid 1a).
BBa_K5401005	T7 RNA polymerase (T7RNAP) antisense oligonucleotide (ASO)	Antisense Oligonucleotide (ASO) system is designed to bind to specific RNA sequences, effectively suppressing the translation of target proteins. By silencing mRNA, ASOs reduce the expression of genes.	Ordered Gblocks based on the designed sequences, cloned into T7RNAP plasmid.
BBa_K5401008	RNAPAnc119	Ancestral reconstruction strands are utilised to infer the evolutionary history and functional characteristics of genes. Allowing for the prediction of ancestral states and the identification of key mutations that may have influenced gene function over time. Which may be beneficial to the evolution of T7RNAP.	Ordered Gblocks based on the designed sequences, cloned into T7RNAP plasmid.
BBa_K5401009	RNAPAnc137	Ancestral reconstruction strands are utilised to infer the evolutionary history and functional characteristics of genes. Allowing for the prediction of ancestral states and the identification of key mutations that may have influenced gene function over time. Which may be beneficial to the evolution of T7RNAP.	Ordered Gblocks based on the designed sequences, cloned into T7RNAP plasmid.
BBa_K5401010	RNAPAnc302 Blast250	Ancestral reconstruction strands are utilised to infer the evolutionary history and functional characteristics of genes. Allowing for the prediction of ancestral states and the identification of key mutations that may have influenced gene function over time. Which may be beneficial to the evolution of T7RNAP.	Ordered Gblocks based on the designed sequences, cloned into T7RNAP plasmid.