Latest revision as of 20:18, 21 October 2019

theophylline riboswitch with optimized RBS + eGFP

This is a part that can test the function of the optimized RBS. The theophylline riboswitch is initially designed by Wieland, M. and Hartig J. S. (2008). We optimize this sequence by replacing the Shine-Dalgarno (SD) sequence with our calculated RBS by thermodynamic model. This switch is theophylline responsive which means this switch will undergo self-cleavage at the presence of theophylline. After self-cleavaging, ribosomes can bind with mRNA and translation will initiate. We use eGFP(BBa_E0040) as our reporter. The fluorescence intensity/OD. 600 can indicate the functionality of this switch. We place this part at the downstream of pBAD promoter. With the induce of L-arabinose, BL21(DE3) cells will transcribe mRNAs containing theophylline switch and eGFP coding sequence. At the presence of theophylline, eGFP protein will be translated and by detecting the fluorescence intensity/OD. 600, the functionality of this switch can be characterized.

Part Inprovement

In this part of experiment. We Characterized our newly designed BBa_k3030016 and BBa_k3030017. In order to get a more significant results, we improved the protocols used by Peking_R 2011 team, these improvements were well-documented in our protocols. We focused on investigating the following points in our relevant experiments:

-The cytotoxicity induced by theophylline to DE3 cells.

-The absorbance of our cultured DE3 in different concentrations of theophylline in our experiments.

-The fluorescence intensity measured that indicates the sensitivity of riboswitch to theophylline.

1.The growth curve of DE3 in different concentrations of theophylline.

We used four gradients of TPP (0 mM, 0.1 mM, 1 mM and 10 mM) to test if the addition of TPP may induce cytotoxicity to DE3, and influence the growth of them. Each single colony of the DE3 cells transfected with circularized pSB1A3 were cultured in 100 ul LB-0.1%Amp for 12.5 hours, the OD600 values were measured in each 15 minutes. We chose 12 colonies in this experiment, the cultured time was compared with the average calculated for the 12 colonies’ OD600. The growth curves of DE3 cells is displayed below:

The growth curve indicates that the OD600 measured may be influenced by the concentration of TPP, and the DE3 incubated with 1 mM TPP grows best and the 10 mM TPP may significantly inhibit the growth of DE3 cells. But the difference in OD600 was not that remarkable when the incubating time is limited in 25000 seconds. Thus, we chose to culture the DE3 cells for 6 hours in the following experiments.

2. Measuring the fluorescence intensity of DE3 cells transformed with standards and blank.

We repeated the Peking_R 2011 experiment and adapted their arrangement of TPP gradients (0 mM without arabinose added, 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM with 1 mM arabinose added). In addition, The absorbance measured was used in normalize the fluorescence intensity measured to minimize the interference caused by bacteria concentration, while the high number of colonies grew in each well may lead a wrong over-high fluorescence result. The absorbance is measured after the GFP fluorescence.

All the procedures were conducted under dark condition in a 96-well plate. The distribution of the colonies cultured is displayed as below:

(The part number of XJTLU-Optimized, PKU-Natural is BBa_k3030017, BBa_598009 respectively)

The GFP fluorescence values were measured, the excitation and emission light was 485 nm and 535 nm respectively. To avoid the leakage of fluorescence interfering the reading of other wells, the time interval of measurement was extended to 20 ms. The exact GFP fluorescent intensity of each well is listed below:

Figure 1: The dose responsive curves that manifesting GFP intensity measured at 535 nm divided by OD600 comparing to the TPP concentration (0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM), the concentration of arabinose in each well was controlled to be 1 mM. It can be shown that the GFP fluorescence intensity increases along with the concentration of TPP as the riboswitch was opened by the TPP.

Figure 2: The GFP/OD600 measured comparing to TPP concentration in each well. Student’s t-test is applied with the figure (*: p<0.05, **:p<0.01, ***:p<0.001).

Figure 3: The working curves that indicates the sensitivity of the riboswitch designed to TPP. The activation ratio is fluorescence intensity under given TPP concentrations compared to that of without TPP. The TPP gradients were set as 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 5 mM, and 10 mM, the concentration of arabinose in each well was controlled to be 1 mM.

Figure 4: The activation ratio measured comparing to TPP concentration in each well. Student’s t-test is applied with the figure (*: p<0.05, **:p<0.01, ***:p<0.001).

The results shows that the riboswitch designed in part BBa_k3030017 has a 10-time expression of GFP (normalized by OD600) comparing to that of BBa_k598009, our improved part. In addition, the designed BBa_k3030017 has a 11-fold activation ratio of the absence to presence of 10mM theophylline.

Reference

Wieland, M., Hartig, J.S. (2008). ‘Improved aptazyme design and in vivo screening enable riboswitching in bacteria.’ Angewandte Chemie, 47(14), pp2604-2607. [Online]. Available from: https://www.ncbi.nlm.nih.gov/pubmed/18270990

Sequence and Features