Parts

“The whole is more than the sum of its parts" - Aristotle

All parts used for IBDetection to be successful can be found on these pages. Part Overview gives a brief overview of all parts used in the sensor-reporter system and the Part Collection explains our used and designed collection in more detail, which is separated in the Basic Parts page and the Composite Parts page. Furthermore, the button Improvement brings you to the page of our improved part.

Part Overview Part Collection Basic Parts Composite Parts Improvement

Part Overview

For our designed system the important Basic Parts of our Composite Parts system were separately tested. These components are all codon-optimized for expression in E. coli and the plasmids were ordered via Addgene. Furthermore, the designed DNA constructs (BBa_K3972003 & BBa_K3972004) were used to combine the sensing part (BBa_K3972000 & BBa_K3972001) and the reporter part (BBa_K3972002), which were both codon-optimized for expression in E. coli and synthesized by IDT. Together, our part collection gives a detailed overview of the complete tetrathionate sensing two-component system TtrS/R, followed by the formation of ultrasound measurable ARG1 gas vesicles. Our favorite parts from this part collection are basic part ARG1 (BBa_K3972002), composite part TetR-pLtetO-1-TtrR-pTtrB185-269-ARG1 (BBa_K3972004) and composite part 5’UTR-RFP (BBa_K3972005).

Part Table

Part Number	Type	Name	Description	Length	Favorite
BBa_K3972000	Basic	TtrS	TtrS is the first component of the two-component sensing system TtrS/R and functions as the membrane-bound sensor kinase (SK), which can sense tetrathionate outside the cell [1].	2016 bp
BBa_K3972001	Basic	TtrR	TtrR is the second component of the two-component sensing system TtrS/R and functions as the DNA-binding response regulator (RR) that binds to the TttrB185-269 promoter (pTtrB185-269) (BBa_K2507019), which can induce gene expression [1].	621 bp
BBa_K3972002	Basic	ARG1	ARG1 is a collection of 12 proteins that together form gas vesicles when expressed. The proteins involved are GvpA, GvpC, GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU [2].	6685 bp	❤
BBa_K3972003	Composite	pTtrB185-269-ARG1	This composite part is a combination of the parts BBa_K2507019 and BBa_K3972002.	6776 bp
BBa_K3972004	Composite	TetR-pLtetO-1-TtrR-pTtrB185-269-ARG1	This composite part is a combination of the part BBa_K3972001 and the part BBa_K3972002. Additionally, functional parts are added to the construct: BBa_K1475003, BBa_K3332034, and BBa_K2507019.	7492 bp	❤
BBa_K3972005	Composite	5’UTR-RFP	This part is an improved part of the RFP Coding Device BBa_K801100, which is improved by the replacement of the RBS BBa_B0034 by a translation enhancing 5’UTR containing a g10-L RBS BBa_K3972006.	1089 bp	❤
BBa_K3972006	Basic	5’UTR with g10 RBS	This part is used to improve the protein expression of part BBa_K801100. The part consists of translation-enhancing DNA, a poly-A-spacer, an RBS, and an AT-rich region.	54 bp
BBa_K2507019	Basic	pTtrB185-269	pTtrB185-269 is a minimal TtrR activated promoter and is activated when TtrR is phosphorylated by TtrS after binding of tetrathionate to TtrS.	85 bp
BBa_I746916	Basic	sfGFP	This part codes for superfolder GFP, which is a folding enhanced GFP and shows improved folding kinetics in E. coli, compared to wild type GFP [3].	720 bp
BBa_J06504	Basic	mCherry	mCherry is a constitutively expressed fluorescent protein and is used for sfGFP signal normalization [4].	711 bp
BBa_K1475003	Basic	TetR	This part expressed as TetR protein. This protein represses the pTet promoter family and is inhibited by tetracycline and analogs (e.g. doxycycline).	627 bp
BBa_K3633015	Basic	pT7	The T7 promoter is a commonly used promoter, which recognizes T7 RNA polymerase and is induced by IPTG [5].	19 bp
BBa_K3332034	Basic	pLtetO-1	This promoter allows the continuous expression of LacI and is repressed by tetR. In the presence of anhydrotetracycline, tetR will be repressed and the pLTetO-1 promoter will be active.	99 bp

References

Daeffler, K., Galley, J., Sheth, R., Ortiz‐Velez, L., Bibb, C., Shroyer, N., Britton, R. and Tabor, J., 2017. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, [online] 13(4), p.923.
Bourdeau R, Lee-Gosselin A, Lakshmanan A, Farhadi A, Kumar S, Nety S et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature. 2018;553(7686):86-90.
Pédelacq J-D, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006;24(1):79–88.
mCherry fluorescent protein [Internet]. Takarabio.com. [cited 2021 Oct 13]. Available from: https://www.takarabio.com/products/gene-function/fluorescent-proteins/fluorescent-protein-plasmids/red-fluorescent-proteins/mcherry-fluorescent-protein
Sigmaaldrich.com. [cited 2021 Oct 13]. Available from: https://www.sigmaaldrich.com/NL/en/technical-documents/technical-article/genomics/cloning-and-expression/t7-promoter-system

Team:TU-Eindhoven/Parts

Part Overview

Part Table

References