Difference between revisions of "Part:BBa K2904092"

Latest revision as of 11:18, 21 October 2019

sfGFP regulated by modular Four U riboswitch

Design

Background of 2019 OUC-China's project——RiboLego

Due to context-dependent performance and limited dynamic range, the widespread application of riboswitches is currently restricted. By replacing its original ORF with a new one, the structure of an aptamer domain can be subtly disrupted, resulting in a loss of ligand response. So riboswitch is still not be considered as a ‘plug and play' device. To tackle these problems, our project focuses on a standardized design principle to be used for modular and tunable riboswitch. The modular riboswitch we defined consists of the original riboswitch, Stabilizer and Tuner. Stabilizer can protect the structure of riboswitch from damage while Tuner can reduce the expression probability of fusion protein and make improvement of riboswitch function.

The construction of this part

Riboswitches can furthermore be classified into thermodynamic and kinetic switches. Different from kinetic switches, thermodynamic switches can reversibly and repeatedly toggle between "on-" and "off-" states, depending on temperature. Thermodynamic switches are temperature-sensing RNA sequences in 5’UTR of their mRNAs. At low temperature, they can fold into the structure, blocking access of ribosome; at high temperature, it open conformation, increasing the efficiency of translation initiation.

After successfully creating modular riboswitch with our method on kinetic switches and demonstrate that Tuner can control the expression of a range of functional outputs, we started to build modular thermodynamic riboswitch to test the universality of our design principle. Four U is an RNA thermometer that can be used for temperature sensitive post-transcriptional regulation that initiates translation at 37°C. Fortunately, we found that OUC-China team had used Four U to successfully express RFP in 2015, which provided us with great convenience! So we selected the first 132bp of RFP as Stabilizer of Four U. Then we used Tuner A to change the exprssion level. The superfolder green fluorescent protein (sfGFP) is the reporter gene to verify our modular riboswitch.

Result

To validate this part containing a new thermodynamic switch, we used a constitutive promoter(BBa_J23119). After construction, the circuit was transformed into DH5αZ1. Given that the temperature threshold of Four U is 37℃, we set the culture temperature to 28℃, 37℃ and 42℃ in liquid LB medium respectively. We can observe a noticeable difference when cultured for 30 hours. Though centrifugation, the recombination strain at 37°C and 42°C shows green while the strain at 28°C shows no fluorescence.

Figure1: The results of liquid test show the working effect of modular Four U in different temperature.

The above qualitative experiments are not enough to verify the effect of modular Four U, so we tested the fluorescence intensity by microplate reader to measure the expression level of the reporter protein legulated by FourU element as temperature-controlled switch. We still set three culture temperatures: 28°C, 30°C and 37°C. After overnight culture, we choose 5 time points (8h, 24h, 30h, 36h, 48h) to test fluorescence intensity of sfGFP protein. The results demonstrate that moduar FourU element is a useful thermodynamic riboswitch and works very well!

Figure2: The results of microplate reader show the working effect of modular Four U element in different temperature.

Sequence and Features