Part:BBa_K1614011

Spinach in BBF RFC 110 transcription cassette

The Spinach II aptamer in RFC 110 cloning standard for in vitro transcription and in vivo expression. With this part we validated the functionality of our new BBF RFC 110 in vitro and in vivo (File:BBF RFC 110.pdf)

Usage and Biology

The aim of the RFC 110 is to perform transcriptions of any RNA of interest and to cleave specifically its 3’end by a ribozyme (HDV). This RFC was applied in in vivo and in vitro assays to perform in vitro transcription of an RNA of interest (Spinach2) which is processed at its 3’-end by a hepatitis delta virus (HDV) ribozyme. To analyze this construct we performed in vitro transcription reaction first. To do so, the insert, containing a T7 RNA polymerase promoter, Spinach2 Aptamer (ROI) and HDV ribozyme was amplified by PCR using the RFC plasmid as template. To determine the correct functioning of the construct (T7-ROI-HDV) we applied the Spinach2 Aptamer as our RNA of interest. If the T7 promoter and the HDV ribozyme act as expected, the Spinach2 aptamer will be transcribed during in vitro transcription reaction. We will perform this reaction in presence of the DFHBI dye, which interacts with the Spinach2 RNA thereby emitting a fluorescent signal. For the in vitro transcription we applied the RFC 110 plasmid as well as the PCR product and measured the fluorescence at 500 nm in real-time similar to our Malachite Green approach shown in the real-time SMS project. Transcription was analyzed by denaturing polyacrylamide gel electrophoresis (PAGE). We were able to determine the successful cleavage of the HDV from the construct by PAGE. To prove the correct functioning of the RFC110 in vivo we transformed the plasmid into E. coli Bl21 (DE3) competent cells. The transcription of our RNA of interest as well as the HDV ribozyme was initiated by addition of IPTG during the exponential growth of the culture. The cells were incubated in presence of DFHBI for 30 min and fixed with poly-L-lysine. The bacteria with the RFC110 containing the Spinach as RNA of interest were analyzed by microscopy. Fluorescence was measured on a GFP channel. We were able to identify fluorescent E. coli if the T7-ROI-HDV was transcribed in vivo. Thus the RFC 110 enables us the transcription of a RNA of interest by the T7 RNA polymerase from a plasmid. The attachment of and HDV virus results in a RNA of interest with a specific 3’-end.

Figure 1.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
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 113
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 113
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 113
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 113
  Illegal NgoMIV site found at 126
  Illegal NgoMIV site found at 155
  
• 1000
  COMPATIBLE WITH RFC[1000]