Engineering bacteria to solve the Burnt Pancake Problem

<p>Abstract</p> <p>Background</p> <p>We investigated the possibility of executing DNA-based computation in living cells by engineering <it>Escherichia coli </it>to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA.</p> <p>Results</p> <p>Inversions (or "flips") of the DNA fragment pancakes are driven by the <it>Salmonella typhimurium </it>Hin/<it>hix </it>DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in <it>E. coli</it>. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; <it>E. coli </it>cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of <it>in vivo </it>flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes.</p> <p>Conclusion</p> <p>The Hin/<it>hix </it>system is a proof-of-concept demonstration of <it>in vivo </it>computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/<it>hix </it>may provide a flexible new tool for manipulating transgenic DNA <it>in vivo</it>.</p

Short Score Certificates for Upset Tournaments

A score certificate for a tournament, T , is a collection of arcs of T which can be uniquely completed to a tournament with the same scorelist as T &apos;s, and the score certificate number of T is the least number of arcs in a score certificate of T . Upper bounds on the score certificate number of upset tournaments are derived. The upset tournaments on n vertices are in one--to--one correspondence with the ordered partitions of n-3, and are &quot;almost&quot; transitive tournaments. For each upset tournament on n vertices a general construction of a score certificate with at most 2n - 3 arcs is given. Also, for the upset tournament, T # , corresponding to the ordered partition #, a score certificate with at most n+2k+ 3 arcs is constructed, where k is the number of parts of # of size at least 2. Lower bounds on the score certificate number of T # in the case that each part is su#ciently large are derived. In particular, the score certificate number of the so-called nearly transitive tournament ..

Attempted use of PACE for riboswitch discovery generates three new translational theophylline riboswitch side products

Abstract Objective The purpose of this project was to use an in vivo method to discover riboswitches that are activated by new ligands. We employed phage-assisted continuous evolution (PACE) to evolve new riboswitches in vivo. We started with one translational riboswitch and one transcriptional riboswitch, both of which were activated by theophylline. We used xanthine as the new target ligand during positive selection followed by negative selection using theophylline. The goal was to generate very large M13 phage populations that contained unknown mutations, some of which would result in new aptamer specificity. We discovered side products of three new theophylline translational riboswitches with different levels of protein production. Results We used next generation sequencing to identify M13 phage that carried riboswitch mutations. We cloned and characterized the most abundant riboswitch mutants and discovered three variants that produce different levels of translational output while retaining their theophylline specificity. Although we were unable to demonstrate evolution of new riboswitch ligand specificity using PACE, we recommend careful design of recombinant M13 phage to avoid evolution of “cheaters” that short circuit the intended selection pressure

Origins of Replication Determine Plasmid Copy Number.

<p>The origins of replication used in the study are listed with their descriptions and part numbers in the Registry of Standard Biological Parts. The means and standard deviations of PCN values were determined by qPCR and yields of minipreps.</p