Part:BBa_K1645999

Dual Color Plasmid (RFP and GFP)

This part is a composite of an RFP gene and a GFP gene from the previously made BioBricks: BBa_I20260 and BBa_J04450, respectively.

It can be used in CRISPRi experiments to demonstrate the effectiveness of sgRNA-Cas9 targeting. It can also be used for other gene expression suppression systems to provide quantitative feedback and data.

It has been characterized using flow cytometry to demonstrate the expression of both RFP and GFP proteins.

Further characterization of this part being used in a CRISPRi system can be found with the BBa_K1645998 BioBrick.

Characterization

Figure 1: A Comparison of GFP and RFP expression from the Dual Colour Plasmid

We performed a simple experiment to demonstrate that both genes in this part are properly expressed. We aim to compare their relative fluorescence to each other, and demonstrate their usability in a more complex system with CRISPRi (found in the characterization of BBa_K1645999.

Methods and Materials

To produce the data, we inoculated the the appropriate E. coli culture into LB and grew it for 4hr to an OD600 of 0.4, followed by induction with IPTG at a final concentration of 1mM for 6hr. We diluted the culture four-fold into chilled formalin (1X PBS, 4% formaldehyde, 1.5% methanol). We then ran a sample and detected fluorescence using an excitation laser wavelength of 488nm at 200mW, as well as SSC at 1.5mW. After acquiring data from 20'000 cells in all channels, we performed analysis on the IDEAS Application v.6 software.

Results and Discussion

In Figure 1. we use flow cytometry (Aminis ImageStream MKII) to show the relative fluorescence of the GFP and RFP proteins.

Figure 1: A Comparison of GFP and RFP expression from the Dual Colour Plasmid

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1708
  Illegal AgeI site found at 1820
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 706