A split intein T7 RNA polymerase for transcriptional AND-logic

Synthetic biology has developed numerous parts for building synthetic gene circuits. However, few parts have been described for prokaryotes to integrate two signals at a promoter in an AND fashion, i.e. the promoter is only activated in the presence of both signals. Here we present a new part for this function: a split intein T7 RNA polymerase. We divide T7 RNA polymerase into two expression domains and fuse each to a split intein. Only when both domains are expressed does the split intein mediate protein trans-splicing, yielding a full-length T7 RNA polymerase that can transcribe genes via a T7 promoter. We demonstrate an AND gate with the new part: the signal-to-background ratio is very high, resulting in an almost digital signal. This has utility for more complex circuits and so we construct a band-pass filter in Escherichia coli. The split intein approach should be widely applicable for engineering artificial gene circuit parts.     

Factors influencing the recombinational expansion and spread of telomeric tandem arrays in Kluyveromyces lactis

We have previously shown that DNA circles containing telomeric repeats and a marker gene can promote the recombinational elongation of telomeres in Kluyveromyces lactis by a mechanism proposed to involve rolling-circle DNA synthesis. Wild-type cells acquire a long tandem array at a single telomere, while telomerase deletion (ter1-Δ) cells, acquire an array and also spread it to multiple telomeres. In this study, we further examine the factors that affect the formation and spread of telomeric tandem arrays. We show that a telomerase⁺ strain with short telomeres and high levels of subtelomeric gene conversion can efficiently form and spread arrays, while a telomere fusion mutant is not efficient at either process. This indicates that an elevated level of gene conversion near telomeres is required for spreading but that growth senescence and a tendency to elongate telomeres in the absence of exogenously added circles are not. Surprisingly, telomeric repeats are frequently deleted from a transforming URA3-telomere circle at or prior to the time of array formation by a mechanism dependent upon the presence of subtelomeric DNA in the circle. We further show that in a ter1-Δ strain, long tandem arrays can arise from telomeres initially containing a single-copy insert of the URA3-telomere sequence. However, the reduced rate of array formation in such strains suggests that single-copy inserts are not typical intermediates in arrays formed from URA3-telomere circles. Using heteroduplex circles, we have demonstrated that either strand of a URA3-telomere circle can be utilized to form telomeric tandem arrays. Consistent with this, we demonstrate that 100-nucleotide single-stranded telomeric circles of either strand can promote recombinational telomere elongation.