1. dcpf1 protein: BBa_K3820006 original dCpf1 is an efficient tool for multiplex gene regulation. In the research, connecting 24nt guide sequences targeting three independent segments within the coding region of the sf-GFP gene by the 36nt DR sequences and co-expressed under a constitutive promoter (A), the researchers found that crRNAs targeting any one of the three segments resulted in varied but significant gene repression(10–100 fold). Repression was further augmented by doubly or triplycombined crRNAs, presumably through a stronger blockage of transcription elongation (C). Strikingly, the triply combined crRNAs completely abolished GFP expression (>300-fold reduction). The fold reduction by multiplex targeting, relative to individual targeting, was between additive and multiplicative. These results suggested that co-transcribed crRNAs targeting multiple DNA segments can be utilized by dCpf1 to combinatorially augment gene repression.
Resource: Chensi Miao, Huiwei Zhao, Long Qian, Chunbo Lou, Systematically investigating the key features of the DNase deactivated Cpf1 for tunable transcription regulation in prokaryotic cells, Synthetic and Systems Biotechnology, Volume 4, Issue 1, 2019, Pages 1-9, ISSN 2405-805X,
2. Vc2 Riboswitch: BBa_K3286202 Vc2 Riboswitch resides upstream of the gene (VC1722) homologous to tfoX of V. Cholerae. Fig1 shows the crystal structure of three-helix Vc2 Riboswitch. Two terminal ends of base-paired regions(P2 and P3, respectively shown in light blue and green) form a third helix(P1, shown in black-blue). c-di-GMP is shown in orange.
Figure 2: Crytsalline structure of vc2 riboswitch with c-di-GMP
Vc2 RNA tends to form a one-to-one saturable complex with a dissociation constant (KD) of ~1 nM for c-di-GMP. As shown by Fig2, Various analogs of c-di-GMP are tested, including the linear breakdown products pGpG, GpG, and GpGpG etc. and results have shown that they are strongly discriminated against by Vc2 aptamer. Moreover, it has an affinity of ~10 pM, making it the tightest binding c-di-GMP receptor and the highest-affinity RNA-ligand interaction known.
Figure 3: Data of Vc2 riboswitch