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Team:OUC-China/RNA guardian/Results
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| - | <div class="span9"><p style="font-weight:normal;"><font size="2px">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:<br /><br /><img src="https://static.igem.org/mediawiki/2013/4/45/Ouc-RNA-result | + | <div class="span9"><p style="font-weight:normal;"><font size="2px">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:<br /><br /><img src="https://static.igem.org/mediawiki/2013/4/45/Ouc-RNA-result.jpg" height="500" width="600" /><br /><br />Table.1 Comparison of the experiment and control groups by measuring the relative fluorescence units (RFU).<br />We measure the fluorescence twice with an interval of 1h. The relative increasing are calculated by this formula: (RFUexperiment-RFUcontrol)/ RFUcontrol.<br /><br />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.<br /><br />But, it is not direct and we only perform two experiments because it is so different to lend a microplate reader. Next, when we come back to school, we will perform RT-PCR for further experiment and provide another evidence.<br /><br />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 it’s sequence in experiment 1. And in experiment 4, there is a obvious decreasing of the RFU, we think may be the extra ribosome effect the binding efficiency of the real ribosome.<br><br>Why the fluorescence of some groups lower? Here we give some reasons and possible solutions.<br><br>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 ensure the RBS is in a single strand region. However, we have no idea if it will be enough intervals between two ribosomes when mRNA forms a secondary structure and how to solve it.<br>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 inquiry further. |
Thirdly, we have not detected the mRNA level. We think, may be the mRNA degradation slows down but the translation efficiency goes down. So in the future, we will use RT-PCR for further experiments. | Thirdly, we have not detected the mRNA level. We think, may be the mRNA degradation slows down but the translation efficiency goes down. So in the future, we will use RT-PCR for further experiments. | ||
Revision as of 00:31, 28 September 2013
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 perform two experiments because it is so different to lend a microplate reader. Next, when we come 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 it’s sequence in experiment 1. And in experiment 4, there is a obvious decreasing of the RFU, we think may be the extra ribosome effect the binding efficiency of the real ribosome.
Why 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 ensure the RBS is in a single strand region. However, we have no idea if it 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 inquiry further.
Thirdly, we have not detected the mRNA level. We think, may be the mRNA degradation slows down but the translation efficiency goes down. So in the future, we will use RT-PCR for further experiments.
