Qiafilter plasmid kit

To construct M13ms9 (8 kb), the 0.42 kb oriC fragment was PCR-amplified from the MG1655 genome using primers SUE260/SUE261, followed by NcoI–NsiI digestion and cloning into a vector prepared from M13mp18 by PCR with primers SUE226/SUE227, followed by NcoI–NsiI digestion. To construct M13ms10 (8 kb), a 0.25 kb fragment containing the chromosomal terB region and a second inverted terB sequence was PCR-amplified from the MG1655 genome using primers SUE236/SUE238, followed by NheI–NsiI digestion and cloning into the XbaI–PstI sites of M13ms9. Duplex, replicative forms of phage were prepared in E. coli JM109 strain and purified using the QIAfilter plasmid kit (Qiagen).
pPKOZ (8.9 kb) was constructed using the cell-free cloning method described in Figure 6. After transformation of DH5α, the plasmid was purified with the QIAfilter plasmid kit. To construct pETcocoKm (11.3 kb), the 1.2 bp Km fragment was PCR-amplified from pUC4k using primers SUE296/SUE375, digested with PciI–PstI, and cloned into the PciI–NsiI sites of pETcoco-2 (Novagen). To construct pETcocoKmOri, a 1 kb oriC fragment was PCR-amplified from the MG1655 genome using primers SUE505/SUE506 and cloned into the NheI–AatII sites of pETcocoKm. Primer sequences are listed in Supplementary Table S1.

Luciferase reporter constructs were generated by introducing the ANG-1 3′-UTR, carrying a putative miR-204/211 binding site, into the vector pGL3 control (Promega, Madison, WI, USA). The 3′-UTR sequence was amplified by PCR using the primers ANG-1-UTR-Sense (5′-CGGGGTACCGCGCAATGTCAGAAGCGATTATG-3′) and ANG-1-UTR-Antisense (5′-GGAAGATCTGTAGTTTGAAGCACAGCAAGC-3′) to introduce KpnI and BglII restriction sites (underlined). The PCR product was cloned into pGL3 control and the resultant plasmid (pGL3C-WT) was used as a template to produce the mutant plasmid pGL3C-MUT. Site-directed mutagenesis of the miR-204/211 binding site was performed using the Quik-Change Site-Directed Mutagenesis kit (Stratagene, Heidelberg, Germany). Specifically, the bases AAA, complementary to UUU in the seed sequence (UUUCCCUU) of miR-204/211, were mutated to TTT; the three base pairs were mutated without introducing or removing other nucleotides in the binding site. All plasmid DNA was purified using the QIAfilter plasmid kit (Qiagen, Hilden, Germany) and confirmed to have the correct sequence by sequencing (Takara, Dalian, China). miR-NC (empty vector), miR-204 (miRBase accession number: MI0000247) and miR-211 (miRBase accession number: MI0000708) overexpressing retroviral vector carrying were constructed as described by Huang et al (19 (link),20 (link)).