RNA-OUT的无抗生素选择系统-RNA-OUT based antibiotic-free selection system

在插入序列10 (IS10) 系统中，RNA-OUT RNA 与RNA-IN RNA 杂交，并减少下游IS10 转座酶基因的翻译。 从质粒中设计出 pLac 表达的 RNA-OUT，该质粒微弱地抑制单拷贝整合 pR-RNA-IN-lacZ 构建体的表达。 虽然这表明RNA-IN、RNA-OUT系统可用于获得异源染色体基因的一定水平的调节，但它并未证明使用该系统进行质粒选择和维持具有足够的抑制作用。

In the insertion sequence 10 (IS10) system, RNA-OUT RNA hybridizes to the RNA-IN RNA, and reduces translation of the downstream IS10 transposase gene . Jaenecke and colleagues reported engineering a pLac-expressed RNA-OUT from a plasmid that weakly repressed expression of a single copy integrated pR-RNA-IN-lacZ construct . While this demonstrates that the RNA-IN, RNA-OUT system can be used to obtain some level of regulation of a heterologous chromosomal gene, it does not demonstrate sufficient repression for use of this system for plasmid selection and maintenance. We report herein adaptation of RNA-OUT, RNA-IN regulation to plasmid selection as follows.

A counter-selectable marker (sacB; Bacillus subtilis levansucrase ) expressed under the control of the RNA-IN promoter and leader was integrated into the chromosome of DH5α (RNA-IN-SacB cell line). Expression of sacB was determined to be constitutive since cells containing sacB encoded levansucrase were killed in the presence of sucrose. Translation of SacB from this RNA was designed to be repressed in the presence of the antisense regulator RNA-OUT . RNA-OUT was cloned into the pDNAVACCUltra-P5/6,4/6_-RBS-EGFP vector, in place of the kanR gene, by replacement of the DraIII-KpnI cassette. pDNAVACCUltra- P5/6,4/6_-RBS-EGFP (RNA-OUT) repressed expression of the RNA-IN regulated chromosomally integrated counter-selectable marker SacB. This allowed antibiotic free plasmid selection in the presence of sucrose on solid and liquid media .

pDNAVACCUltra- P5/6,4/6_-RBS-EGFP (RNA-OUT)

A fluorescent RNA-OUT plasmid was created. The pDNAVACCUltra- P5/6,5/6_-RBS-EGFP plasmid (expresses EGFP constitutively) was digested with DraIII/KpnI and the linear vector (2029, 1015) purified. The following 2 RNA-OUT primers were annealed. These produce an annealed RNA-OUT fragment that is compatible with DraIII and KpnI .

RNA-OUTF01: 5′ gtggtagaattggtaaagagagtcgtgtaaaatatcgagttcgcacatcttgttgtctgattattgatttttggcgaaaccatttgatcatatg acaagatgtgtatctaccttaacttaatgattttgataaaaatcattaggtac 3′

RNA-OUTR01: 5′ ctaatgatttttatcaaaatcattaagttaaggtagatacacatcttgtcatatgatcaaatggtttcgccaaaaatcaataatcagacaacaa gatgtgcgaactcgatattttacacgactctctttaccaattctaccacaac 3′

The annealed primers were ligated into the vector, and transformed into the RNA-IN-sacB integrated cell line from step 2.3 above. Colonies were selected on LB (no NaCl) +6% sucrose and plasmid from two fluorescent colonies were sequence verified as correct. Transformation of sequence-verified pDNAVACCUltra-P5/6,4/6_-RBS-EGFP (RNA-OUT) plasmid into RNA-IN –SacB competent cells was performed; all recovered colonies after sucrose selection were fluorescent (i.e. 100% of recovered cells contained the plasmid).