Difference between revisions of "Part:BBa K1615089"

Latest revision as of 23:24, 21 September 2015

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.

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



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



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.



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.



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