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Team:HokkaidoU Japan/RBS/Conclusion
From 2013.igem.org
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| - | <h1 id="common-header-title">Maestro E.coli</h1> | + | <h1 id="common-header-title">Maestro <span class="italic">E. coli</span></h1> |
<h2 id="common-header-subtitle">RBS</h2> | <h2 id="common-header-subtitle">RBS</h2> | ||
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Preciously SD6 was reported as the strongest. However our results indicated SD4. | Preciously SD6 was reported as the strongest. However our results indicated SD4. | ||
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<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"> | <img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"> | ||
| + | <div style="padding-bottom: 0;"><span class="bold">fig.1 The difference of expression level. </span>If there is A/U rich enhancer, SD sequence has mighty effect.</div> | ||
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| + | <div style="padding-bottom: 0;"><span class="bold">fig.2 LacZα expression level. </span>Difference of SD sequence gives different amount of LacZα expression.</div> | ||
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The sequence we synthesized was completely the same. | The sequence we synthesized was completely the same. | ||
Latest revision as of 03:57, 29 October 2013
Maestro E. coli
RBS
Conclusion
We were successful at making four RBS set with four strength levels. Construct with SD4 showed the strongest β-Galactosidase activity. Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.
Though we synthesized the RBSs based on previous reports, we got unexpected results. Preciously SD6 was reported as the strongest. However our results indicated SD4.
The sequence we synthesized was completely the same. Both plasmids had low copy number. One thing we changed was the reporter gene. In the previous experiments, GFP was used for the reporter. In contrast, we used LacZα. The expected strength differed only by changing the coding sequence.
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life. Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency. Kudla et al. (2009) showed that translation efficiency is determined by factors in Coding-Sequence. Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.
Our results show different levels of translation efficiency. We should try and repeat our assays with other reporter genes. The translation efficiency might change by choosing different coding sequences. The translation efficiency not depended on RBS but also influenced by coding sequence. This fact complicates designing biological devices.
When regulating the expression it is important to have variability in RBSs strength. Our set RBSs proved to have different efficiencies in both GFP and LacZα assays. From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site. Therefore, we made a useful set of RBSs for regulating expression.
- Grzegorz Kudla, et al. Coding-Sequence Determinants of Gene Expression in Escherichia coli (2009) Science
