Connecting Boolean Integrase Logic gates to a novel alkane control signal via engineered level matching.

Boolean Integrase Logic gates use phage integrases that respond to transcriptional signals to implement logical functions within living cells via DNA recombination. Control of biological systems using these logic gates has many applications, including biomanufacturing, healthcare, or environmental remediation. Gates are highly sensitive to background transcriptional noise producing unwanted integrase expression and uncontrolled, permanent gate switching. Consequently, connecting gates to novel control signals requires time-consuming directed evolution of regions regulating integrase expression. Here we present an approach in which the activity of an alkane biosensor is tuned to match levels of existing integrase control signals. By adjusting AlkS expression, we tuned the transcriptional output of Palkb to match the output of existing integrase controllers. We successfully connected Palkb to two integrases with different transcriptional control requirements and demonstrated the original logic function was conserved. Our method complements directed evolution approaches to connect Boolean integrate logic gates to novel transcriptional sources and will ultimately facilitate the systematic in silico design of gates responding to various control signals.

Comparative Genomic Analysis and Benzene, Toluene, Ethylbenzene, ando-,m-, andp-Xylene (BTEX) Degradation Pathways of Pseudoxanthomonas spadix BD-a59

ABSTRACTPseudoxanthomonas spadixBD-a59, isolated from gasoline-contaminated soil, has the ability to degrade all six BTEX (benzene, toluene, ethylbenzene, ando-,m-, andp-xylene) compounds. The genomic features of strain BD-a59 were analyzed bioinformatically and compared with those of another fully sequencedPseudoxanthomonasstrain,P. suwonensis11-1, which was isolated from cotton waste compost. The genome of strain BD-a59 differed from that of strain 11-1 in many characteristics, including the number of rRNA operons, dioxygenases, monooxygenases, genomic islands (GIs), and heavy metal resistance genes. A high abundance of phage integrases and GIs and the patterns in several other genetic measures (e.g., GC content, GC skew, Karlin signature, and clustered regularly interspaced short palindromic repeat [CRISPR] gene homology) indicated that strain BD-a59's genomic architecture may have been altered through horizontal gene transfers (HGT), phage attack, and genetic reshuffling during its evolutionary history. The genes for benzene/toluene, ethylbenzene, and xylene degradations were encoded on GI-9, -13, and -21, respectively, which suggests that they may have been acquired by HGT. We used bioinformatics to predict the biodegradation pathways of the six BTEX compounds, and these pathways were proved experimentally through the analysis of the intermediates of each BTEX compound using a gas chromatograph and mass spectrometry (GC-MS). The elevated abundances of dioxygenases, monooxygenases, and rRNA operons in strain BD-a59 (relative to strain 11-1), as well as other genomic characteristics, likely confer traits that enhance ecological fitness by enabling strain BD-a59 to degrade hydrocarbons in the soil environment.

Comparative Genome Analysis of “Candidatus Phytoplasma australiense” (Subgroup tuf-Australia I; rp-A) and “Ca. Phytoplasma asteris” Strains OY-M and AY-WB

ABSTRACT The chromosome sequence of “Candidatus Phytoplasma australiense” (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. Potential mobile units (PMUs) containing clusters of DNA repeats comprised 12.1% of the genome. These PMUs encoded genes involved in DNA replication, repair, and recombination; nucleotide transport and metabolism; translation; and ribosomal structure. Elements with similarities to phage integrases found in these mobile units were difficult to classify, as they were similar to both insertion sequences and bacteriophages. Comparative analysis of “Ca. Phytoplasma australiense” with “Ca. Phytoplasma asteris” strains OY-M and AY-WB showed that the gene order was more conserved between the closely related “Ca. Phytoplasma asteris” strains than to “Ca. Phytoplasma australiense.” Differences observed between “Ca. Phytoplasma australiense” and “Ca. Phytoplasma asteris” strains included the chromosome size (18,693 bp larger than OY-M), a larger number of genes with assigned function, and hypothetical proteins with unknown function.

Targeting site-specific chromosome integration.

The concept of gene therapy was introduced with great promise and high expectations. However, what appeared simple in theory has not translated into practice. Despite some success in clinical trials, the research community is still facing an old problem: namely, the need for a vector that can deliver a gene to target cells without adverse events while maintaining a long-term therapeutic effect. Some of these challenges are being addressed by the development of hybrid vectors which meld two different viral systems to incorporate efficient gene delivery and large cloning capacity with site-specific integration. The two known systems that integrate genes into specific sites in mammalian genomes are the adeno-associated virus and phage integrases. Recent experiments with hybrid vectors incorporating both of these systems are encouraging. However, extensive research should be directed towards the safety and efficacy of this approach before it will be available for gene therapy.