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

Figure 2: Frequency of GFP and RFP Intensity Measurements

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 appropriate E. coli culture into LB and grew it for 12hr at 37 degrees Celsius on a shaker at 200RPM. We diluted the culture four-fold into chilled formalin (1X PBS, 4% formaldehyde, 1.5% methanol). We used flow cytometry (Aminis ImageStream MKII) to run 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.

This protocol is based off in-house protocols created by previous Waterloo iGEM members and revised over the years by advisors and experienced users.

Results and Discussion

Figure 1. shows that both RFP and GFP are fluorescing, though at different intensities. Overall, GFP's intensity data averages at 502 intensity units and RFP's intensity data averages at 139 intensity units. This means that GFP fluoresces approximately 3.5x more intensely than RFP. Figure 2 shows the frequency at which cells fluoresce at a particular intensity for GFP on the right and RFP on the left.

In all, further experiments can provide more precise measurements of GFP and RFP fluorescence, but we present here adequate fluorescence data for other teams to understand the behavior of the Dual Colour Plasmid in a DH5α chassis.

Caveats and Considerations

In other characterization experiments detailed with BBa_K1645999, we realized the RFP's promoter (BBa_R0010) has a LacI binding site meaning that expression of our Dual Colour Plasmid in a chassis that produces the lac repressor will require induction with IPTG or lactose. For induction protocols with the this part, please refer to the details found with BBa_K1645999.

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