Team:OUC-China/Results
TEST PREVIOUS STEM-LOOPS
It is reported that there are several native stem-loops that have effects on its flanking genes, either at the 3’ end or the 5’ end[1]. Ergo, we use two native stem-loops from R. capsulatus and E.coli[2] and a previously experimented stem-loops with different free energy to preliminary verify that stem-loops in the intergenic region can regulate the relative expression of two reporter genes within polycistrons. We measured them on both transcriptional and translational level.
The result see as follows:
Figure 1 Both relative expression on RNA and protein level with stem-loop of -25.6 kcal/mol (measured by Mfold) are significantly higher than those of control group without stem-loop, which means stem-loops can coordinate the expression within cistrons. The vertical coordinate is the ratio of upstream gene gfp to downstream gene mcherry. Error bars indicate s.d. of mean of experiments in triplicate.
After the function and sequences of stem-loops confirmed, we standardized and submitted them to iGEM part registry with detailed description.
Click here to see more.
Results suggested that stem-loops at 3’ end functioned well in regulation. Hence we tested another two native stem-loops in the following.
Figure 2 Structures of three previous stem-loops
Figure 3 Stem-loops with lower folding free energy lead to stronger protection of upstream gene, which results in higher ratio of GFP to mCherry both in transcriptional and translational level. Error bars indicate s.d. of mean of experiments in triplicate
MEASURE A SERIES OF DESIGNED STEM-LOOPS
In addition to the confirmation of previous stem-loops, we also devoted to exploring new stem-loops designed by ourselves. It is difficult to estimate the relationship between structure and energy, thus we weighed several software and finally chose Mfold, a commonly acknowledged one. Employing the same rules, we designed several stem-loops (Figure 4) with various free folding energy and tested them (Figure 5). As shows, these parts tune the ratio of the upstream and downstream genes as well.
Figure 4 Structure of three stem-loops designed by ourselves
Both relative expression on RNA and protein level of designed stem-loops of -30.1, -34.4,38.8,44.9kcal/mol (measured by Mfold) compare to the control group without stem-loop. The vertical coordinates is the ratio of upstream gfp to downstream mCherry. Error bars indicate s.d. of mean of experiments in triplicate.
CONSTRUCT A WONDERFUL TOOLKIT
Collecting those validated previous stem-loops and our designed ones, we got a set of stem-loops to form a toolkit. We measured them both in transcriptional and translational level. We have submitted these parts to iGEM parts registry with detailed characterization. With the demands of bigger library and more detailed data, we are working on more stem-loops to expand our toolkit library.
Figure 4 Structure of three stem-loops designed by ourselves
FURTHER EXPLORATION OF FREE ENERGY
We measured them both on transcriptional and translational level. We have submitted these parts to iGEM parts registry with detailed characterization. And with the demands of bigger library and more detailed data, we are working on more stem-loops to expand our toolkit library. Below is the relative expression on two levels of different stem-loops in our toolkit.
Figure 6 RNA varying with folding free energy of stem-loops
Figure 7 protein expression varying with folding free energy of stem-loop
There may be certain relationship between the relative expression and free energy, so we did more to explore the relationship, see details at model.
Cistrons Concerto
Thanks
1.Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences
2.NEW ENGLAND Biolabs
3.GenScript
Contact us:
E-mail: oucigem@163.com
Designed and built by @ Jasmine Chen and @ Zexin Jiao
We are OUC-iGEM
