O'Keefe, S. | Search bioRxiv

bioRxiv2019

Synthetic cassettes for pH-mediated sensing, counting and containment

AbstractAs pH is fundamental to all biological processes, pH-responsive bacterial genetic circuits enable precise sensing in any environment. Where unintentional release of engineered bacteria poses a concern, coupling pH sensing to expression of a toxin creates an effective bacterial containment system. Here, we present a pH-sensitive kill switch (acidic Termination of Replicating Population; acidTRP), based on the E. coli asr promoter, with a survival ratio of less than 1 in 106. We integrate acidTRP with cryodeath to produce a two-factor containment system with a combined survival ratio of less than 1 in 1011 whilst maintaining evolutionary stability. We further develop a pulse-counting circuit with single cell readout for each administered stimulus pulse. We use this pulse-counter to record multiple pH changes and combine it with acidTRP to make a two-count acid-sensitive kill switch. These results demonstrate the ability to build complex genetic systems for biological containment.

synthetic biology↗

bioRxiv2019

Skin colonization by circulating neoplastic clones in cutaneous T-cell lymphoma

Mycosis fungoides (MF) is a mature T-cell lymphoma currently thought to develop primarily in the skin by a clonal expansion of a transformed, resident memory T-cell. However, this concept does not explain the key characteristics of MF such as the debut with multiple, widespread skin lesions or inability of skin directed therapies to provide cure. The testable inference of the mature T-cell theory is the clonality of MF with respect to all rearranged T-cell receptor (TCR) genes. Here we have used whole exome sequencing approach to detect and quantify TCR, -{beta} and -{gamma} clonotypes in tumor cell clusters microdissected from MF lesions. This method allows us to calculate the tumor cell fraction of the sample and therefore an unequivocal identification of the TCR clonotypes as neoplastic. Analysis of TCR sequences from 29 patients with MF stage I-IV proved existence of multiple T-cell clones within the tumor cell fraction, with a considerable variation between patients and between lesions from the same patient (median 11 clones, range 2-80 clones/sample). We have also detected multiple neoplastic clones in the peripheral blood in all examined patients. Based on these findings we propose that circulating neoplastic T-cell clones continuously replenish the lesions of MF thus increasing their heterogeneity by a mechanism analogous to the consecutive tumor seeding. We hypothesize that circulating neoplastic clones might be a promising target for therapy and could be exploited as a potential biomarker in MF.

cancer biology↗