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).
|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 .||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 .||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 .||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 .||720 bp|
|BBa_J06504||Basic||mCherry||mCherry is a constitutively expressed fluorescent protein and is used for sfGFP signal normalization .||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 .||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|
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