MIT's software enables easier codon optimization, pairwise sequence alignment, diagnostic digests, and primer design for CAR-TK parts.
Prior to ordering custom DNA sequences intended for transfection, it is imperative for teams to optimize codons for the host organism. Specifically, codon optimization refers to the process of changing codons without changing their encoded amino acids in order to accommodate the codon bias inherent to the host organism. This increases both gene expression and translational efficiency of the gene of interest.
Though some online tools exist to aid in codon optimization, our team faced significant uncertainty and confusion while trying to utilize them, and results were frequently irreproducible and did not reflect expectations based on codon frequency charts. Thus, we decided to create software that accurately optimizes sequences with consistent results. We have implemented optimization for many frequently used host organisms, including homo sapiens, mus musculus, and many more. We hope that our tool will provide a straightforward method for future iGEM teams and other researchers to optimize their codons.
Sequence alignment is central to biological experimentation, with extensive applications enabling everything from inference of evolutionary relationships between genes or proteins to comparison of synthetic genetic constructs with their theoretical designs. Some software include alignment tools as auxiliary and suboptimally designed resources, and computational packages like BioPython provide sufficient functionality for experienced coders to align their own sequences; however, there are no easily accessible and straightforward online alignment tools where users can simply upload their sequences and view alignment information.
We experienced these difficulties firsthand when we received sequencing results of some of our most important plasmid constructs and found that aligning them with our theoretical designs was frustrating and confusing. Thus, we implemented our own pairwise sequence alignment tool.
Modern cloning methods are excellent and typically result in correctly constructed plasmids, but errors are always possible, and it is crucial to verify constructs before using them for experiments. Sequencing is a foolproof method of verifying constructs, but costs can rapidly accumulate. Thus, for daily lab activity, our team often turns to enzymatic digestion of plasmids followed by agarose gel electrophoresis to roughly determine whether plasmids appear correct.
Planning these crucial diagnostic digests can be tedious and prone to human error. To mitigate these issues, our team developed a user-friendly tool that makes planning digests quick and effortless.
Click here to use our diagnostic digest aid!
To facilitate researchers' usage of our Chimeric Antigen Receptor assembly ToolKit (CAR-TK), we have provided software that automatically designs primers for PCRing out fragments of constructed CAR plasmids, in accordance with the assembly standards we proposed in our CAR-TK design. More information, usage examples, and download options are available on our GitHub repository.