Difference between revisions of "Part:BBa K2310000"

Revision as of 15:56, 31 October 2017

Bio-signal amplifier based on T7 system

To solve the problem that the detection limit of the existing method to detect the metal ions in water is so high, so we designed a bio-amplifier to strengthen the signal provided by the sensing device.

T7 amplification system is the main feature of the amplifier system. The E.coli T7 system is regarded as the most widely used system for high-level gene expression. This system consists of a lambda DE3 lysogenic E.coli strain carrying a chromosomally integrated copy of the T7 RNA polymerase gene (gene 1) controlled by the lacUV5 promoter and a high-copy number vector allowing target gene expression from the T7 promoter. In contrast to other E.coli expression systems using host RNA polymerases for heterologous gene expression, an appropriate T7 system yields higher protein amounts since the bacteriophage RNA polymerase exhibits enhanced processivity.

T7 RNA polymerase is highly selective for its own promoters, which do not exist naturally in Escherichia coli. A relatively small number of T7 RNA polymerases provided from a cloned copy of T7 gene is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E.coli RNA polymerase is greatly decreased.

This part, an intermediate to construct our detector, is the amplifier to strengthen the intensity of bio-signals.

Usage and Biology

In our experiment, for testing the amplification effect of our circuit, we use GFP, BBa_E0040 as the reporter, because the fluorescence intensity is easier to be measured. Also, the intensity of the fluorescence can stand for the intensity of the corresponding genes' expression, and the data of the fluorescence intensity could be more convenient for us to model the circuit. Meanwhile, we chose BBa_J33201, an arsenic induced promoter, as our sensor to detect arsenic in water samples.

The amplifier test system (Experimental group)	The no-amplified system (Control group)

We designed a double-plasmid system, which contains BBa_K2310009 and BBa_K2310002 as Figure 1 shows, and a control group that only contains a GFP expression system with the same promoter that can be induced by arsenic as Figure 2 shows. Both the two systems were transformed into E.coli DH5α and incubated with arsenious acid standard solution (the concentration of arsenic is 0, 2.6, 6.5 and 97.5μM) for 24 hours. After that, we measured OD600 of the E.coli and the fluorescence intensity of GFP for 900 minutes. The OD600 showed the growth trend of the engineering bacteria and the fluorescence intensity of each group showed the relationship between arsenic and the expression of our circuits.

To ensure that the concentration of arsenic would not influence the growth of the bacteria, we incubated our bacteria with adding arsenious acid standard solution into the medium.

Figure 3 The OD600 versus time of E.coli with different concentration of arsenic

From Figure 3, we found that the value of OD600 after incubating for 24 hours is about 0.55, and the growth curves of each group don't show much difference. It suggested that they had the same growth trend, so the difference of fluorescence intensity was mainly from the expression of the circuits or other factors.

Sequence and Features

Experiments

We did experiments at the same condition to measure the fluorescence intensity of GFP. As showed in Figure 4 with the increase of arsenic, the expression of the GFP showed an upper trend. In addition, the green fluorescence intensity of the amplified group is much higher than that of the unamplified groups, about 1.5 times higher.

Figure 4 The average green fluorescence intensity versus time of each group

These show that the amplification effect of our circuit is effective and all right, and our system works well.