Journal: Cell Reports

Article Title: Optogenetic Tuning Reveals Rho Amplification-Dependent Dynamics of a Cell Contraction Signal Network

doi: 10.1016/j.celrep.2020.108467

Figure Lengend Snippet:

Article Snippet: For pTriEx-NTOM20-CCL-moxBFP-CCL-LOV2 the mVenus fluorophore from pTriEx-NTOM20-mVenus-LOV2 (kind gift of Klaus Hahn, UNC-Chapel Hill School of Medicine, ) was replaced by CCL-moxBFP-CCL (CCL: coiled-coil) by PCR amplification of the backbone with primers 5′-ttctgagcagcggttccggatccggtTCCTTGGCTACTACACTTG-3′ and 5′-TCTAGATTTAAAGTTCGGATCG-3′ and moxBFP ( ) (Addgene Plasmid 68064) including CCL-domains with priners 5′-atccgaactttaaatctagaggatctggtagtggttccGCTAGCCTCGCAGCTGCG-3′ and 5′-TCCGGAACCGCTGCTCAG-3′, followed by Gibson assembly. mTurquoise2-eDHFR-GEF-H1 (C53R) was generated by PCR amplification of the mTurquoise2-NES-eDHFR backbone from mTurquoise2-NES-eDHFR-RhoA Q63L ΔCAAX using primers 5′-CTCAATTGTTGTTGTTAACTTG-3′ and 5′-GGATCTAGAGGTGGATCCCCG-3′ and PCR amplification of GEF-H1 C53R including a N-terminal linker region from mCherry-Zdk1-GEF-H1 C53R using primers 5′-ggcggggatccacctctagatccGGTGGTTCTGGTGGTAGC-3′ and 5′-agttaacaacaacaattgagTTAGCTCTCGGAGGCTAC-3′ followed by Gibson assembly. mTurqouise2-eDHFR-LARG was generated by replacing GEF-H1 C53R from mTurquoise2-eDHFR-GEF-H1 C53R with LARG, using NotI and MfeI restriction sites and PCR amplification of LARG from mCherry-LARG (kind gift of Oliver Rocks, MDC Berlin) with primers 5′-aaaatctgtatttccagggcggccgctctggaagtggaAGTGGCACACAGTCTACTATC-3′ and 5′-ataaacaagttaacaacaacTCAACTTTTATCTGAGTGCTTG-3′, followed by Gibson assembly.

Techniques: Virus, Recombinant, Plasmid Preparation, Software