Team:TU-Eindhoven/Basic Parts
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.
Basic Parts
Three different basic parts have been characterized and optimized for the application in our designed Composite Parts. The following basic parts have been tested separately as described on Proof of Concept.
TtrS (BBa_K3972000) and TtrR (BBa_K3972001)
The E. coli codon-optimized basic parts TtrS and TtrR are both derived from the sensing two-component system of the marine bacterium Shewanella Baltica. TtrS is the first component of this system and functions as the transmembrane-bound sensor kinase (SK). The kinase can bind tetrathionate extracellularly with high specificity. The second component, TtrR, is the DNA-binding response regulator (RR) that is activated by TtrS and subsequently binds to pTtrB185-269 (BBa_K2507019) as a phosphorylated dimer. This dimer induces gene expression and thereby induces GFP expression [1] (Figure 1).
Figure 1: Overview TtrS/R system.
To characterize the activation and subsequent behavior of the TtrS/R system, a superfold GFP protein (BBa_I746916) was expressed under the regulation of pTtrB185-269. The IPTG inducible promoter Ptac (BBa_K2558004) regulates TtrS, while TtrR is regulated by a leaky anhydrotetracycline inducible promoter pLtetO-1 (BBa_K3332034). Furthermore, constitutively expressed mCherry (BBa_J06504) is present in the TtrR plasmid, of which the signal is used for normalization of the sfGFP signal. For more details on this check Results segment TtrS/R sensing system. An overview of all components of the TtrS/R plasmids can be seen in Figure 2.
Figure 2: TtrS/R plasmids.
ARG1 (BBa_K3972002)
E. coli codon-optimized ARG1 codes for a collection of 12 gas vesicle proteins (Gvp) which assemble into gas vesicles upon expression. The involved proteins are GvpA, GvpC, GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU. The ARG1 gene consists of two repeats of the GvpA protein and four repeats of the GvpC protein, in contrast to all other Gvps, which only contain one repeat. GvpA and GvpC are derived from the cyanobacteria species Aphanizomenon flos-aquae and form the fundamental structure and scaffold for the gas vesicles by dimerizing into a biconical shape. The other proteins are derived from Bacillus Megaterium and assist in the formation and stability of the gas vesicles by acting as chaperone proteins [2,3].
The ARG1 gene is under control of the IPTG inducible T7 promoter (BBa_K3633015) (Figure 4) and after protein expression gas vesicles are formed, as shown in Figure 3. These gas vesicles produce contrast in ultrasound images, due to the ability of the proteins to scatter the incoming sound waves of the ultrasound transducer. Using acoustic pulses with an amplitude above the critical collapse pressure, the gas vesicles can be collapsed [2]. Images can be taken before and after the collapse, and image-analysis can be performed to obtain clear images of the number of gas vesicles present inside of the bacteria. Examples of such ultrasound images can be found in Results segment ARG1 reporter system.
Figure 3: Overview ARG1 system.
Figure 4: ARG1 plasmid.
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 | ❤ |
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Daeffler, K. N., Galley, J. D., Sheth, R. U., Ortiz-Velez, L. C., Bibb, C. O., Shroyer, N. F., Britton, R. A., & Tabor, J. J. (2017). Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular systems biology, 13(4), 923. https://doi.org/10.15252/msb.20167416
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Bourdeau, R., Lee-Gosselin, A., Lakshmanan, A. et al. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature 553, 86–90. https://doi.org/10.1038/nature25021
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Pfeifer, F. (2012). Distribution, formation and regulation of gas vesicles. Nat Rev Microbiol 10, 705–715. https://doi.org/10.1038/nrmicro2834