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.

Visualization of evolutionary stability dynamics and competitive fitness of Escherichia coli engineered with randomized multigene circuits.

Strain engineering for synthetic biology and metabolic engineering applications often requires the expression of foreign proteins that can reduce cellular fitness. In order to quantify and visualize the evolutionary stability dynamics in engineered populations of Escherichia coli , we constructed randomized CMY (cyan-magenta-yellow) genetic circuits with independently randomized promoters, ribosome binding sites, and transcriptional terminators that express cyan fluorescent protein (CFP), red fluorescent protein (RFP), and yellow fluorescent protein (YFP). Using a CMY color system allows for a spectrum of different colors to be produced under UV light since the relative ratio of fluorescent proteins vary between circuits, and this system can be used for the visualization of evolutionary stability dynamics. Evolutionary stability results from 192 evolved populations (24 CMY circuits with 8 replicates each) indicate that both the number of repeated sequences and overall expression levels contribute to circuit stability. The most evolutionarily robust circuit has no repeated parts, lower expression levels, and is about 3-fold more stable relative to a rationally designed circuit. Visualization results show that evolutionary dynamics are highly stochastic between replicate evolved populations and color changes over evolutionary time are consistent with quantitative data. We also measured the competitive fitness of different mutants in an evolved population and find that fitness is highest in mutants that express a lower number of genes (0 and 1 > 2 > 3). In addition, we find that individual circuits with expression levels below 10% of the maximum have significantly higher evolutionary stability, suggesting there may be a hypothetical "fitness threshold" that can be used for robust circuit design.

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.

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.

Computational tools for metabolic engineering.

A great variety of software applications are now employed in the metabolic engineering field. These applications have been created to support a wide range of experimental and analysis techniques. Computational tools are utilized throughout the metabolic engineering workflow to extract and interpret relevant information from large data sets, to present complex models in a more manageable form, and to propose efficient network design strategies. In this review, we present a number of tools that can assist in modifying and understanding cellular metabolic networks. The review covers seven areas of relevance to metabolic engineers. These include metabolic reconstruction efforts, network visualization, nucleic acid and protein engineering, metabolic flux analysis, pathway prospecting, post-structural network analysis and culture optimization. The list of available tools is extensive and we can only highlight a small, representative portion of the tools from each area.

Designing and engineering evolutionary robust genetic circuits.

BACKGROUND: One 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. RESULTS: We 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. CONCLUSIONS: A 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.

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.

Genetic basis of evolutionary adaptation by Escherichia coli to stressful cycles of freezing, thawing and growth.

Microbial evolution experiments offer a powerful approach for coupling changes in complex phenotypes, including fitness and its components, with specific mutations. Here we investigate mutations substituted in 15 lines of Escherichia coli that evolved for 1000 generations under freeze-thaw-growth (FTG) conditions. To investigate the genetic basis of their improvements, we screened many of the lines for mutations involving insertion sequence (IS) elements and identified two genes where multiple lines had similar mutations. Three lines had IS150 insertions in cls, which encodes cardiolipin synthase, and 8 lines had IS150 insertions in the uspA-uspB intergenic region, encoding two universal stress proteins. Another line had an 11-bp deletion mutation in the cls gene. Strain reconstructions and competitions demonstrated that this deletion is beneficial under the FTG regime in its evolved genetic background. Further experiments showed that this cls mutation helps maintain membrane fluidity after freezing and thawing and improves freeze-thaw (FT) survival. Reconstruction of isogenic strains also showed that the IS150 insertions in uspA/B are beneficial under the FTG regime. The evolved insertions reduce uspB transcription and increase both FT survival and recovery, but the physiological mechanism for this fitness improvement remains unknown.

Evolutionary adaptation to freeze-thaw-growth cycles in Escherichia coli.

Fifteen populations of Escherichia coli were propagated for 150 freeze-thaw-growth (FTG) cycles in order to study the phenotypic and genetic changes that evolve under these stressful conditions. Here we present the phenotypic differences between the evolved lines and their progenitors as measured by competition experiments and growth curves. Three FTG lines evolved from an ancestral strain that was previously used to start a long-term evolution experiment, while the other 12 FTG lines are derived from clones that had previously evolved for 20,000 generations at constant 37 degrees C. Competition experiments indicate that the former FTG group improved their mean fitness under the FTG regime by about 90% relative to their progenitor, while the latter FTG group gained on average about 60% relative to their own progenitors. These increases in fitness result from both improved survival during freezing and thawing and more rapid recovery to initiate exponential growth after thawing. This shorter lag phase is specific to recovery after freezing and thawing. Future work will seek to identify the mutations responsible for evolutionary adaptation to the FTG environment and use them to explore the physiological mechanisms that allow increased survival and more rapid recovery.

Increased susceptibility to repeated freeze-thaw cycles in Escherichia coli following long-term evolution in a benign environment.

BACKGROUND: In order to study the dynamics of evolutionary change, 12 populations of E. coli B were serially propagated for 20,000 generations in minimal glucose medium at constant 37 degrees C. Correlated changes in various other traits have been previously associated with the improvement in competitive fitness in the selective environment. This study examines whether these evolved lines changed in their ability to tolerate the stresses of prolonged freezing and repeated freeze-thaw cycles during adaptation to a benign environment. RESULTS: All 12 lines that evolved in the benign environment for 20,000 generations are more sensitive to freeze-thaw cycles than their ancestor. The evolved lines have an average mortality rate of 54% per daily cycle, compared to the ancestral rate of 34%. By contrast, there was no significant difference between the evolved lines and their ancestor in mortality during prolonged freezing. There was also some variability among the evolved lines in susceptibility to repeated freeze-thaw cycles. Those lines that had evolved higher competitive fitness in the minimal glucose medium at 37 degrees C also had higher mortality during freeze-thaw cycles. This variability was not associated, however, with differences among lines in DNA repair functionality and mutability. CONCLUSION: The consistency of the evolutionary declines in freeze-thaw tolerance, the correlation between fitness in glucose medium at 37 degrees C and mortality during freeze-thaw cycles, and the absence of greater declines in freeze-thaw survival among the hypermutable lines all indicate a trade-off between performance in minimal glucose medium at 37 degrees C and the capacity to tolerate this stress. Analyses of the mutations that enhance fitness at 37 degrees C may shed light on the physiological basis of this trade-off.