Coding
mRFP

Part:BBa_K1615089

Designed by: Brooke Rothschild-Mancinelli   Group: iGEM15_Edinburgh   (2015-08-31)

mRFP RFC25

RFP is important in synthetic biology as a visualisation tool. While multiple versions of RFP exist in the registry, only one is RFC25 compatible. This RFP (BBa_K1351021) contains a Shine-Dalgarno sequence in the RFC25 prefix which precludes it from using the common lac expression cassette (BBa_K314103) which already contains a ribosome binding site and only produces RFC10 fusions. By adding the RFC25 prefix without the Shine-Dalgarno sequence we hope to improve the utility of this RFP.
We visualised RFP using multiple methods to show that it was folding and expressing.

RFP plate.jpg



This plate shows our RFP under the control of the lac expression cassette from iGEM10_Washington (BBa_K314103).

RFP pellet.jpg

When grown in 10 mL of LB + chloramphenicol and spun down, a pink pellet can be visualised.

BBa K1615089 lysate.jpg

To ensure the fluorescence of our RFP we aliquoted 200 µL of the crude cell lysate into a 96 well plate and visualised it under blue light transilluminator. As you can see, it fluoresces consistently in all three samples.

BBa K1615089 saturation.jpg

We wanted to test to see how long it would take to fully saturate an ISO standard Whatman 54 paper chad with RFP. This graph shows that the chad is saturated almost immediately.

BBa K1615089.jpg

We tested the dissociation of this part on Whatman 54 using isostandard chads. This involved incubating the chads in the crude cell lysate for 10 minutes and then washing them with PBS for various periods time to test the binding affinity of this CBD. The results show a gradual loss of fluorescence indicating the RFP leaving the paper. 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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/reporter/rfp
//function/reporter/fluorescence
Parameters
colorRed