Sean C. Sleight

In-Fusion BioBrick assembly and re-engineering

Genetic circuits can be assembled from standardized biological parts called BioBricks. Examples of BioBricks include promoters, ribosome-binding sites, coding sequences and transcriptional terminators. Standard BioBrick assembly normally involves restriction enzyme digestion and ligation of two BioBricks at a time. The method described here is an alternative assembly strategy that allows for two or more PCR-amplified BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit. This method allows for a large number of parallel assemblies to be performed and is a flexible way to mix and match BioBricks. In-Fusion assembly can be semi-standardized by the use of simple primer design rules that minimize the time involved in planning assembly reactions. We describe the success rate and mutation rate of In-Fusion assembled genetic circuits using various homology and primer lengths. We also demonstrate the success and flexibility of this method with six specific examples of BioBrick assembly and re-engineering. These examples include assembly of two basic parts, part swapping, a deletion, an insertion, and three-way In-Fusion assemblies.

Designing and engineering evolutionary robust genetic circuits

BackgroundOne problem with engineered genetic circuits in synthetic microbes is their stability over evolutionary time in the absence of selective pressure. Since design of a selective environment for maintaining function of a circuit will be unique to every circuit, general design principles are needed for engineering evolutionary robust circuits that permit the long-term study or applied use of synthetic circuits.ResultsWe first measured the stability of two BioBrick-assembled genetic circuits propagated in Escherichia coli over multiple generations and the mutations that caused their loss-of-function. The first circuit, T9002, loses function in less than 20 generations and the mutation that repeatedly causes its loss-of-function is a deletion between two homologous transcriptional terminators. To measure the effect between transcriptional terminator homology levels and evolutionary stability, we re-engineered six versions of T9002 with a different transcriptional terminator at the end of the circuit. When there is no homology between terminators, the evolutionary half-life of this circuit is significantly improved over 2-fold and is independent of the expression level. Removing homology between terminators and decreasing expression level 4-fold increases the evolutionary half-life over 17-fold. The second circuit, I7101, loses function in less than 50 generations due to a deletion between repeated operator sequences in the promoter. This circuit was re-engineered with different promoters from a promoter library and using a kanamycin resistance gene (kanR) within the circuit to put a selective pressure on the promoter. The evolutionary stability dynamics and loss-of-function mutations in all these circuits are described. We also found that on average, evolutionary half-life exponentially decreases with increasing expression levels.ConclusionsA wide variety of loss-of-function mutations are observed in BioBrick-assembled genetic circuits including point mutations, small insertions and deletions, large deletions, and insertion sequence (IS) element insertions that often occur in the scar sequence between parts. Promoter mutations are selected for more than any other biological part. Genetic circuits can be re-engineered to be more evolutionary robust with a few simple design principles: high expression of genetic circuits comes with the cost of low evolutionary stability, avoid repeated sequences, and the use of inducible promoters increases stability. Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.

Rationally designed bidirectional promoter improves the evolutionary stability of synthetic genetic circuits

One problem with synthetic genes in genetically engineered organisms is that these foreign DNAs will eventually lose their functions over evolutionary time in absence of selective pressures. This general limitation can restrain the long-term study and industrial application of synthetic genetic circuits. Previous studies have shown that because of their crucial regulatory functions, prokaryotic promoters in synthetic genetic circuits are especially vulnerable to mutations. In this study, we rationally designed robust bidirectional promoters (BDPs), which are self-protected through the complementarity of their overlapping forward and backward promoter sequences on DNA duplex. When the transcription of a target non-essential gene (e.g. green fluorescent protein) was coupled to the transcription of an essential gene (e.g. antibiotic resistance gene) through the BDP, the evolutionary half-time of the gene of interest increases 4–10 times, depending on the strain and experimental conditions used. This design of using BDPs to increase the mutational stability of genetic circuits can be potentially applied to synthetic biology applications in general.

Randomized BioBrick Assembly: A Novel DNA Assembly Method for Randomizing and Optimizing Genetic Circuits and Metabolic Pathways

The optimization of genetic circuits and metabolic pathways often involves constructing various iterations of the same construct or using directed evolution to achieve the desired function. Alternatively, a method that randomizes individual parts in the same assembly reaction could be used for optimization by allowing for the ability to screen large numbers of individual clones expressing randomized circuits or pathways for optimal function. Here we describe a new assembly method to randomize genetic circuits and metabolic pathways from modular DNA fragments derived from PCR-amplified BioBricks. As a proof-of-principle for this method, we successfully assembled CMY (Cyan-Magenta-Yellow) three-gene circuits using Gibson Assembly that express CFP, RFP, and YFP with independently randomized promoters, ribosome binding sites, transcriptional terminators, and all parts randomized simultaneously. Sequencing results from 24 CMY circuits with various parts randomized show that 20/24 circuits are distinct and expression varies over a 200-fold range above background levels. We then adapted this method to randomize the same parts with enzyme coding sequences from the lycopene biosynthesis pathway instead of fluorescent proteins, designed to independently express each enzyme in the pathway from a different promoter. Lycopene production is improved using this randomization method by about 30% relative to the highest polycistronic-expressing pathway. These results demonstrate the potential of generating nearly 20,000 unique circuit or pathway combinations when three parts are permutated at each position in a three-gene circuit or pathway, and the methodology can likely be adapted to other circuits and pathways to maximize products of interest.

BioBrick™ assembly using the In-Fusion PCR Cloning Kit.

Synthetic biologists assemble genetic circuits from standardized biological parts called BioBricks™. BioBrick™ examples include promoters, ribosome binding sites, DNA or RNA-coding sequences, and transcriptional terminators. Standard BioBrick™ assembly normally involves assembly of two BioBricks™ at a time using restriction enzymes and DNA ligase. Here we describe an alternative BioBrick™ assembly protocol that describes the assembly of two BioBricks™ using the In-Fusion PCR Cloning Kit. This protocol can also be adapted to use similar recombination-based assembly methods, such as SLIC and Gibson assembly.

Design and construction of a prototype CMY (Cyan-Magenta-Yellow) genetic circuit as a mutational readout device to measure evolutionary stability dynamics and determine design principles for robust synthetic systems

Synthetic biology is the engineering discipline for constructing novel organisms with functions that do not exist in nature. We recently engineered a prototype CMY genetic circuit that visually produces cyan, magenta, and yellow colors independently and in combination using different inducer molecules. Since the production of each color can be independently controlled, this allows for the production of a spectrum of colors that can be visualized in normal light conditions, and each color can be quantified using fluorescence measurements. We performed an evolution experiment to measure the evolutionary stability dynamics of this prototype CMY genetic circuit in 88 replicate populations of Escherichia coli, propagated with all colors turned on. Our results using particular inducer concentrations show that all 88 replicate populations change from a dark, green-brown color to a cyanish color after only 40 generations. In order to visualize the results of this experiment, we washed and concentrated the cells from each population into a different well of a 384-well plate at different evolutionary timepoints. The color change seen visually is confirmed with quantitative data that demonstrates the loss-of-function of both magenta and yellow colors with variation between replicate populations. We sequenced a single clone from four independently evolved populations and all clones have the same loss-of-function deletion mutation between homologous transcriptional terminators that removes the magenta and yellow expression cassettes. This parallel evolution was somewhat expected from results of previous work, but we expect that randomized and re-engineered versions of this circuit without repeats will produce more divergent results due to more stochastic loss-of-function mutations. This prototype CMY circuit serves as a mutational readout device and allows for a colorimetric and quantitative demonstration of evolution in action using synthetic biology.