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Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering.

ABSTRACT: DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable 'scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

SUBMITTER: Taylor GM

PROVIDER: S-EPMC6379671 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering.

Taylor George M GM Mordaka Paweł M PM Heap John T JT

Nucleic acids research 20190201 3

DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable 'scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both f ...[more]

PMID: 30462270

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Project description:BackgroundEffective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and electro-transfection are the most popular strategies for genetic material transduction. Compared with viral-particle-mediated approaches, electro-transfection is theoretically safer, because it does not promote transgene integration into the host genome. Additionally, the simplicity and speed of the procedure increases the attractiveness of electroporation. Here, we developed and optimized an electro-transfection method for the production of engineered chimeric antigen receptor (CAR)-T cells.ResultsStimulation of T cells had the greatest effect on their transfection, with stimulation of cells for up to 3 days substantially improving transfection efficiency. Additionally, the strength of the external electric field, input cell number, and the initial amount of DNA significantly affected transfection performance. The voltage applied during electroporation affected plasmid permeation and was negatively correlated with the number of viable cells after electroporation. Moreover, higher plasmid concentration increased the proportion of positively transfected cells, but decreased cell viability, and for single-activated cells, higher cell density enhanced their viability. We evaluated the effects of two clinically relevant factors, serum supplementation in the culture medium and cryopreservation immediately after the isolation of peripheral blood lymphocytes. Our findings showed that our protocol performed well using xeno-free cultured, fresh T cells, with application resulting in a lower but acceptable transfection efficiency of cells cultured with fetal bovine serum or thawed cells. Furthermore, we described an optimized procedure to generate CAR-T cells within 6 days and that exhibited cytotoxicity toward targeted cells.ConclusionsOur investigation of DNA electro-transfection for the use in human primary T cell engineering established and validated an optimized method for the construction of functional CAR-T cells.

Project description:BackgroundThe aim of this study is to compare the results of stop-start technique with stop-start technique together with sphincter control training applied in the treatment of premature ejaculation.MethodsThis research was conducted as a pre-test post-test quasi-experimental study. The sample of the study consisted of 80 men. The study was conducted on men who applied to the urology outpatient clinic of LIV Hospital, a prıvate hospital, in Gaziantep, Turkey, between 01 October 2021 and 01 March 2022. "Personal Information Form", "Intravaginal Ejaculation Latency Time (IELT)", "Fold Increase Intravaginal Ejaculation Latency Time (F-IELT)" "Premature Ejaculation Diagnostic Tool (PEDT) Questionnaire" and "Arabic Index Premature Ejaculation (AIPE)" were used as the data collection tools. Behavioral therapy, consisting of a total of 6 sessions, was applied once every two weeks, with each session lasting for 45 minutes. After 3rd and 6th months from the beginning of the application, the data collection tools were applied again. "Stop-Start Technique (Group A)" and "Stop-Start Technique and Sphincter Control Training (Group B)" were used in the treatment.ResultsIn both groups, the IELT and AIPE values after 3rd and 6th months from the beginning of the application were statistically higher than those obtained before (p<0.05). IELT and AIPE values increased more in Group B than Group A (p<0.05). F-IELT values after 6th months from the beginning of the application were found to be statistically significant with a low level of effect size than those obtained before (p<0.05, Cohen's d = 0.027). In both groups, the PEDT values in the 3rd and 6th months after the application were statistically lower than those seen before (p<0.05). PEDT value decreased more in Group B than Group A (p<0.05). The differences between the two groups' IELT (Cohen's d = 0.011), AIPE (Cohen's d = 0.044), and PEDT (Cohen's d = 0.066) values in the 3rd month after the application and IELT (Cohen's d = 0.025), AIPE (Cohen's d = 0.048), and PEDT (Cohen's d = 0.024) values in the 6th month after the application were found to be clinically weak.ConclusionsIt was determined that the stop-start technique given to men with premature ejaculation increased the time spent in the vagina and eliminated the problem of premature ejaculation. It was determined that the stop-start technique in combination with sphincter control training were more effective than the stop-start technique alone.

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Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering.

Publications

Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering.

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