Part:BBa_K2967012
The fixed-gain bio-amplifier
The tunable biological amplifier (Fig. 1) comprises three modular terminals--the input, the output and a gain-tuning input. The device can continuously process the input transcriptional signal with an externally tunable gain (the amplification ratio of the changes in output to input) control.[1]
Figure 1. The composition of tunable biological amplifier. Transcription input can be amplified through the amplifier.
At first, we construct the fixed-gain amplifier (Fig. 2) for the prework. In order to reduce the impact of colonization of exotic microorganisms in the intestine and to accelerate the secretion of IL-10 and myrosinase， we found a gain-tunable transcription amplifier in Pseudomonas syringae to tunable amplifier the NO signal.
To verify the fixed-gain amplification(Fig. 2) capability, we integrated the T7 promoter as the nitric oxide responsive transcriptional sensor to the input of the fixed-gain amplifier with GFP as the output. When the transduced transcriptional input from the T7 promoter was connected to our amplifier, the resulting output signal amplitude and dynamic range increased significantly as well as the response sensitivity to the inducer (Fig 3.).
Figure 2. Diagram for fixed-gain amplifier in pCDFDuet-1 plasmid. T7 promoter, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. lacO, the sequence represses the nearby promoter when there is NO inducer (e.g. IPTG). RBS, ribosome binding site. hrpR and hrpS are activator proteins. PhrpL, a promoter which can be induced by the ultrasensitive high-order co-complex hrpRS. GFP, green fluorescent protein.
Figure 3. Responses of the GFP without fixed-gain amplification (V-GFP) and with fixed-gain amplification (V-AM). The cells are induced by 5 varying concentrations of IPTG (0, 10-6 M, 10-5 M, 10-4 M, 10-3 M) after 4 and 6 hours (A, 4h; B, 6h).
Amplifier and GFP are cloned on pcdfDuet-1 vectors which are called V-AM and V-GFP. Then empty plasmid (VE), VE-GFP and VE-AM are transformed into E. coli BL-21, and single colonies are added to 5 ml LB medium for overnight incubation at 37 degrees Celsius. Using 1 ml PBS to wash the bacteria and measuring the value of OD600 to obtain cell concentration of each culture medium. The bacterial culture medium was diluted to OD600=0.025 with LBS (LB with streptomycin) and then added to black 96-well plate. Isopropyl-beta-D-thiogalactopyranoside (IPTG) with concentration of 0, 10-6 M, 10-5 M, 10-4 M and 10-3 M was added to induce those bacteria. After incubation for 4 hours and 6hours, the data were detected by enzyme labeling instrument. The excitation wavelength is 485 nm and the absorption wavelength is 525 nm.
In conclusion, the amplifier achieves the desired effect, and the amplification gain is about 2.0. Comparing other amplifiers, this value is still low. In addition, we find that the gene expression process is accelerated with the amplifier, the report GFP usually reaching its saturation value after about 4 hours' induction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2102
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1312
Illegal BsaI.rc site found at 3047
Illegal SapI.rc site found at 1945
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