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 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 original 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
We chose to improve BBa_K1467400 because the fusion sites for entire transcriptional units were required in order for us to use this part in pSB1A3 as our Golden Gate destination vector.
We were interested in reducing the amount of time between transformation of Golden Gate samples and the appearance of visibly red colonies. The original part BBa_K1467400 has RFP under a LacI-regulated promoter (BBa_R0010) (BBa_R0010) and a medium RBS (BBa_B0034). We replaced these components with the strong constitutive promoter BBa_J23100 and a strong RBS BBa_B0030. We also codon optimized the sequence for high expression in E. coli.
Golden Gate Assembly Reaction Efficiency
In order to test efficiency, Golden Gate assembly reactions were set up so each contained the same amount of sequence-confirmed destination vector (either BBa_K1467400 or BBa_K3114015) and the required DNA inserts in the form of PCR products. The four inserts that were assembled included BBa_K3114012), BBa_K3114001), BBa_K3114007), and BBa_K3114013). A 1:1 insert:destination vector ratio was used in each reaction. They were conducted as per our Golden Gate assembly protocol.
The Golden Gate assembly products were transformed into E. coli DH5-alpha. Some colonies became visibly red under normal light after 12 hours for the improved part BBa_K3114015) and after 18 hours for BBa_K1467400. This indicates that the improvement was successful. We counted the number of red versus white colonies for each of the replicates 24 hours after transformation. The results suggest that using the improved RFP flipper BBa_K3114015) resulted in a higher percentage of successful assembly reactions compared to the original RFP flipper BBa_K1467400. However, the mechanism behind this result is unclear.
Figure 1. Percent of colonies that are white in appearance 24 hours after transformation for Golden Gate assembly reactions using 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).
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