Part:BBa_K1905003
Riboswitch to detect E5 mRNA from HPV-16 and express RFP
DNA coding for a riboswitch to detect E5 mRNA from Human Papillomavirus type 16 and express Red Fluorescent Protein. This part includes a promoter (BB_R0010) and a RBS (BBa_B0034), the reverse complementary for a section of E5 DNA coding region, and the original sequence with one base changed, RFP coding region (BBa_E1010) and a terminator (BBa_B0015). When there is presence of E5 mRNA, the riboswitch turns on and it allows the expression of RFP as reporter protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 838
Illegal AgeI site found at 950 - 1000COMPATIBLE WITH RFC[1000]
This part is based on part BBa_K1905000 (Riboswitch to detect E5 mRNA) with the red fluorescent protein coding sequence. For the practical purposes of our project, we needed a easily detectable result, so we decided to add the coding sequence for RFP, in order that the results could be reared according to color changing. So when E5 mRNA interacts with the riboswitch, there will be expression of RFP.
Figure 1. Biobrick design diagram
This biobrick was chemically synthesised and cloned into both pSB1A3 and pSB1C3 vectors. These were transformed into E. coli TOP10 strain. It was grown at 37°C for 12 hours and then propagated on liquid LB medium. Miniprep plasmid extraction was carried out in order to document the plasmid, as observed on Figure 2.
Figure 2. Agarose (0.8%) gel electrophoresis; Miniprep plasmid extraction. Lane 3: BBa_K1905003 - E5 Riboswitch + RFP ligation
Furthermore, this biobrick worked as initially expected for the techniques that were carried out. White colonies were seen upon plaquing transformed bacteria, as the riboswitch would only allow the expression of reporter protein (Red) only when in contact with the viral mRNA. As we cannot work with this kind of biological material, our characterisation consisted only in observing white colonies vs. coloured colonies (if the riboswitch turned out to be non-functional).
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