Composite

Part:BBa_K1614002

Designed by: Stefan Holderbach   Group: iGEM15_Heidelberg   (2015-09-14)

transcription cassette of BBF RFC 110

Unit for RFC 110 assembly, clonable in RFC 10 vectors like pSB1C3.

Usage and Biology

This part is the core of the RFC we propose (RFC110, File:BBF RFC 110.pdf) for the generation of functional RNA. It comprises a cassette for generating RNAs with well-defined ends, thereby guaranteeing predictable and reproducible functionality (Figure 1). The BioBrick Standard as described by BBF RFC 10 enables the assembly of multiple DNA fragments or modules called parts. Its key advantage is that the assembly of several parts can be performed by the repetitive use of a simple protocol using only a small set of relatively cheap methods and enzymes. Also the limitation to only few restriction enzymes lowered the likelihood for the presence of cut sites in the genes of interest. Yet the use of traditional restriction cloning presents significant disadvantages when it comes to DNA elements where the cloning product has to be seamless, like for protein fusions, and even has to be sequence specific base-by-base. This is the case if the RNA transcript of the sequence is responsible for the function of the part and not a translated protein. As every base may determine the tertiary structure and thereby behavior of the functional RNA, there are no “wobble bases” that allow the easy substitution of a base. This part has been tested in vitro (Fig. 2A and 2B) and in vivo in BL21 E. coli cells by inserting a Spinach2 construct as RNA of interest. RNA was visualized by adding DFHBI to BL21 cells which have been exposed to 100 µM IPTG for 2 hours (Fig. 2C and 2D).

Fig. 1.Suggested workflow proposed in BBF RFC 110. Using standardized DNA constructs being available in the Registry of Biological Parts, any desired functional RNA can be produced in a controlled manner. For simplicity the DNA template that will be transcribed into RNA is considered being the RNA of Interest.
Fig. 2.Validation of the RFC. (A) Real time monitoring of in vitro transcription using the fluorescence of Spinach2. In presence of the DFHBI dye an increase in fluorescence could be monitored for the in vitro transcription using the plasmid as well was the PCR product as template. This proves the functionality of the Spinach transcribed from the biobrick BBa_K1614002. (B) UV-Shadowing shows the self-cleaving function of the HDV-ribozyme leading to two products: HDV RNA and Spinach2 that contains a defined 3’-end (C) Increase in fluorescence could be measured in E. coli BL21(DE3) expressing BBa_K1614002 with Spinach2 as insert. Negative control: Empty plasmid. Background due to autofluorescence of the E. coli cells. (D) Fluorescence microscopy images of cells expressing BBa_K1614002 with and without Spinach2 as insert.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 32
    Illegal NgoMIV site found at 61
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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