Team:Calgary/Contribution
RFP Flipper Devices
Biobricks for red-white colony screening in Golden Gate assembly
RFP Golden Gate flippers consist of an RFP coding device (BBa_J04450) flanked with inverted BsaI sites and fusion sites compatible with the MoClo assembly standard (Weber et al., 2011). They can be used to convert parts designed for Type IIS or MoClo assembly into standard BioBricks. They can also be used to domesticate any BioBrick RFC[10] compatible vector to a Golden Gate destination vector.
Successful assembly reactions that use this part will result in the substitution of the RFP coding device by the Golden Gate inserts. As a result, colonies with successful assembly products can be screened by their white colour. Colonies containing unsuccessful assembly products (ie. the undigested destination vector) will be red in appearance. The reaction scheme is demonstrated below.
Our team discovered four of these RFP flipper devices in the iGEM registry when we were investigating different Golden Gate destination vector candidates. They offered us the opportunity to assemble our genetic circuits in the pSB1C3, pSB1A3, or pSB1K3 vectors that we were most familiar with. The RFP flipper devices in the iGEM registry were as follows:
- BBa_K1467100 - MoClo fusion sites for promoter and 5’UTR sequences
- BBa_K1467200 - MoClo fusion sites for signal peptide and coding sequences
- BBa_K1467300 - MoClo fusion sites for terminator sequences
- BBa_K1467400 - MoClo fusion sites for entire transcriptional units
Characterization
Golden Gate Assembly Reaction Efficiency
As part of our effort to improve BBa_K1467400, we tested the efficiency of Golden Gate assembly reactions using different RFP flipper devices. Reaction were set up so each contained the same amount of sequence-confirmed destination vector (BBa_K1467200 BBa_K1467400, or BBa_K3114015) and the required DNA inserts in the form of PCR products. A 1:1 insert:destination vector ratio was used in each reaction. They were conducted as per our Golden Gate assembly protocol..
As per the overhang/fusion site sequences associated with the BsaI restriction sites in BBa_K3114015 and BBa_K1467400, they were used in four-insert assembly reactions using parts:
The other RFP Golden Gate flipper (BBa_K1467200) features overhang/fusion site sequences associated with the BsaI restriction sites that necessitated two-insert assembly reactions using parts:
The Golden Gate assembly products were transformed into E. coli DH5-alpha. Some colonies became visibly red under normal light after 12 hours for BBa_K3114015, and after 18 hours for BBa_K1467200 and BBa_K1467400. We counted the number of red versus white colonies 24 hours after transformation for each of the replicates. The results suggest that the two-insert Golden Gate assembly reactions had slightly lower efficiency compared to the four-insert reactions for the closely-related BBa_K1467400 part. Further analysis is required to determine whether this is caused by the differences in the flipper device designs or due to sequence elements within the DNA inserts that we used for these experiments. The improved flipper BBa_K3114015 showed higher efficiency than both BBa_K1467400 and BBa_K1467200.
Figure 1. Percent of colonies that are white in appearance 24 hours after transformation for Golden Gate assembly reactions using pSB1A3-BBa_K1467200, pSB1A3-BBa_K1467400, and pSB1A3-BBa_K3114015 as destination vectors. Control reactions were conducted by adding all reagents to the Golden Gate reaction except the DNA inserts. Values represent the mean for three replicates. Error bars indicate standard error of the mean (SEM).
References
Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765
