Part:BBa_K3611009
A cascade transduction signal amplifier
When there are few input signal, the report signal output by the engineered bacteria will be very weak. In order to ensure people’s health and safety as much as possible, we hope the detection limit of our detection system is low enough and the output level is high enough to ensure that trace viruses would not be missed. In order to achieve this, we designed a cascade amplifier to amplify the detected virus signal, to increase the expression of the reporter gene and lower the detection limit.
The cascade amplifier comprises two modular terminals—the input and the output. It consists of two independent orthogonal amplifiers arranged in series, which are constructed using hrp operon from Pseudomonas syringae, and extracytoplasmic function (ECF) σ factor ECF11_987 and promoter ECF11_3726 from Vibrio parahaemolyticus.
The natural hrp regulatory network contains activator protein HrpR and HrpS. These two would form an ultrasensitive high-order co-complex HrpRS, which bind the upstream activator sequence of the hrpL promoter to remodel the closed σ54-RNAP-hrpL transcription complex to an open one through ATP hrdrolysis. This have been proven to amplify the input transcriptional signal.
Bacterial sigma factors (σs), the promoter recognition subunits of RNA polymerase (RNAP), are modular proteins with domains that recognize DNA sequences in the -10 and -35 regions of their target promoters. In addition to the housekeeping σs that recognize the thousands of promoters, bacteria have a variable number of stress activated alternative σs that direct RNAP to distinct promoter sequences. Extracytoplasmic function (ECF) σs are one type of them. By comparison promoters of ECFs are highly conserved, which means they could only be specificity activated by their own corresponding ECFs. The promoters also have a large dynamic range of output, where the OFF state is very low in the absence of the σs and the ON state produces a high level of expression, which could be used as a signal amplify part. We select ECF11_987 σs and promoter ECF11_3726, which are proved as a higher-gain amplifier combination, to build our secondary amplifier.
In principle, the transduced transcriptional signal of the sensor could be sequentially amplified through multilayer coupled amplifiers. We connect two amplifiers in series. The first amplifier’s output is the second amplifier’s input, so that the second amplifier could amplify the first one’s output signal. In general, the initial sensor’s signal will be amplified twice.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1897
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1879
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1089
Illegal SapI.rc site found at 1722
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