Difference between revisions of "Part:BBa K2360003"

Latest revision as of 03:29, 28 October 2017

rrnB' terminator+MerR+pMerR

This part is a MerR family operon which can make a specific reponse to mercury ion. We add a strong terminator in 5', so that we can just add a report module in 3' to make a biosensor.

Sequence and Features

Figure 1. Bio-circuit of BBa_K2360003

Background

MerR

    The MerR family is a group of transcriptional regulators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognized. The few MerR-like regulators that have been studied experimentally have been shown to activate suboptimal σ70-dependent promoters, in which the spacing between the -35 and -10 elements recognized by the σ factor is greater than the optimal 17±1 bp. So, how does it respond to heavy metal ions? Take MerR-pMerR as an example, it has several processes. 1. In the absence of Hg²⁺ and MerR, RNA polymerase preferentially transcribes from the merR promoter, increasing the amount of MerR present in the cell. 2. Once MerR binds to merOP, transcription of the MerR promoter is repressed and the DNA becomes bent and unwound at the operator sequence. RNA polymerase is recruited to the mer promoter, forming a ternary complex of DNA, MerR and RNA polymerase. 3. Binding of Hg²⁺ to one of two binding sites on the MerR make DNA distortion at the centre of the operator which cause the reorientation of the -35 and -10 sequences make them interact with the RNA polymerase σ70 subunit to form an open transcriptional complex and transcription is initiated. [1]

Experiment

Lysis gene expression

We have developed the lysis module (BBa_K2360000) and five metal detecting module for E. coli BL21. Combining the lysis module and detecting module, we have constructed five devices which can respond to different metal ions. Because the lysis gene will be expressed when the detecting module makes a response to the metal ions, which will make bacterial cleavage, resulting in the decrease of OD600, we measured the growth curve of our engineering bacteria in different concentration of metal ions to prove its function.
Figure 1. the growth curve of mercury detecting device in different concentration of mercury ions.
As shown in the growth curves of E. coli with mercury detecting device (figure 2), the growth rate of E. coli BL21 is decline obviously after adding the concentration of Hg²⁺ from 10^-5M to 10^-7M, indicating that this device can detect the mercury ions.

Specificity

We verified the detecting devices specificity of mercury, cadmium and lead by adding Cd²⁺, Hg²⁺, Pb²⁺, Cr⁶⁺, Ni²⁺ to their culture mediums.
figure 1. The growth of the E. coli with mercury detecting device after adding different metal ions and ddH²O