Team:OUC-China/RNA guardian/Results

Our goals are to stabilize the mRNA and as a result, improve the expression of a gene, or rather mamK. We used GFP-LVA as a reporter to examine the expression of a gene. By measuring the fluorescence, we can get the information about how our device worked. Look at this table:



Table.1 Comparison of the experiment and control groups by measuring the relative fluorescence units (RFU).
We measure the fluorescence twice with an interval of 1h. The relative increasing are calculated by this formula: (RFUexperiment-RFUcontrol)/ RFUcontrol.

From this data, we can see, there is obvious improving of the fluorescence, indicating the improving of the expression. Because we use the same RBS and promoter, we think the transcriptional and the translational efficiency are the same. So we think, this is a succinct evidence of the increasing mRNA level.

But, it is not direct and we only did two experiments because it is so different to borrow a microplate reader. Next, when we go back to school, we will perform RT-PCR for further experiment and provide another evidence.

We also have another two experiments groups, experiment 1(both with K1059003 and k1059004), and experiment groups 2 (with part k1059005). But, there’ something wrong with its sequence in experiment 1. And in experiment 4, there is a obvious decreasing of the RFU, we think maybe the extra ribosome has effect on the binding efficiency of the real ribosome.

Why is the fluorescence of some groups lower? Here we give some reasons and possible solutions.

Firstly, because of the extra RBS in the 5’-end, the extra ribosome may inhibit the normal binding of the real ribosome. When we perform our design, in order to prevent the interaction of the two RBSs, we design more than forty nucleotides between the two RBSs. And for ribosome’s efficient binding, we predicted the secondary structure of the part’s transcript, and ensured that the RBS is in a single strand region. However, we have no idea if there will be enough intervals between two ribosomes when mRNA forms a secondary structure and how to solve it.
Secondly, we are not sure whether our mRNA contains digest site of RNaseE because the digest site is not definite. In our future work, we will add extra probable digest site and probe further into the project. Thirdly, we have not detected the mRNA level. We think that maybe the mRNA degradation slows down but at the same time the translation efficiency goes down. So in the future, we will use RT-PCR for further experiments.