Difference between revisions of "Part:BBa K1614002"
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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 (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 (Figure 1). | ||
| + | The RFC was even tested <i> in vivo </i> in BL21 by inserting a Spinach2 construct as RNA of Interest. RNA was visualized by adding DFHBI to BL21 which have been exposed to 100 µM IPTG for 2 hours (Fig. 2C and 2D). | ||
[[File:HeidelbergPWiGEM2015_RFC.png|600 px|thumb|'''Fig. 1.'''General mechanism of the RFC which is based on BioBrick BBa_K1614002]] | [[File:HeidelbergPWiGEM2015_RFC.png|600 px|thumb|'''Fig. 1.'''General mechanism of the RFC which is based on BioBrick BBa_K1614002]] | ||
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Revision as of 16:44, 20 September 2015
transcription cassette of BBF RFC 110
Unit for RFC 110 assembly, clonable in RFC 10 vectors like pSB1C3.
Usage and Biology
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 (Figure 1). The RFC was even tested in vivo in BL21 by inserting a Spinach2 construct as RNA of Interest. RNA was visualized by adding DFHBI to BL21 which have been exposed to 100 µM IPTG for 2 hours (Fig. 2C and 2D).
This part is the core of the RFC we propose for the generation of functional RNA. It comprises a cassette for generating well-defined end of the RNA-sequence, thereby guaranteeing predictable and reproducible functionality.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 32
Illegal NgoMIV site found at 61 - 1000COMPATIBLE WITH RFC[1000]