Synthesis of Libraries and Multi-Site Mutagenesis using a PCR derived, dU-containing Template

Directed DNA libraries are useful because they focus genetic diversity in the most important regions within a sequence. Ideally, all sequences in such libraries should appear with the same frequency and there should be no significant background from the starting sequence. These properties maximize the number of different sequences that can be screened. Described herein is a method termed SLUPT (Synthesis of Libraries via a dU-containing PCR Template) for generating highly targeted DNA libraries and/or multi-site mutations wherein the altered bases may be widely distributed within a target sequence. This method is highly efficient and modular. Moreover, multiple distinct sites, each with one or more base changes, can be altered in a single reaction. There is very low background from the starting sequence, and SLUPT libraries have similar representation of each base at the positions selected for variation. The SLUPT method utilizes a single stranded dU-containing DNA template that is made by PCR. Synthesis of the template in this way is significantly easier than has been described earlier. A series of oligonucleotide primers that are homologous to the template and encode the desired genetic diversity are extended and ligated in a single reaction to form the mutated product sequence or library. After selective inactivation of the template, only the product library is amplified. There are no restrictions on the spacing of the mutagenic primers except that they cannot overlap.

Short Communication: Detection and expression analysis of the bile salt hydrolase gene in Pediococcus and Lactobacillus

Widodo, Nuratri Y, Sukarno AS, Ahyuningsih TD. 2020. Short Communication: Detection and expression analysis of the bile salt hydrolase gene in Pediococcus and Lactobacillus. Biodiversitas 21: 5901-5905. Bile salt hydrolase (BSH) has been considered an important enzyme that is produced in response to bile stress. The bsh gene reportedly encodes BSH in lactic acid bacteria. Thus, the aim of this study was to detect and measure the changes in bsh gene expression in Pediococcus pentosaceus M103, Lactobacillus casei AP, Lactobacillus casei AG, and Lactobacillus paracasei M104. A BSH primer was used in amplifying the bacterial genomic DNA, and the obtained amplicon was then sequenced and analyzed in order to detect genes homologous to bsh. Changes in expression levels were measured in three concentrations of bile salts: 0.1%, 0.3%, and 0.5% (w/v). The BSH primer produced amplicons 850 to 950 kb in length. Amino acid sequences from the amplicons were determined to be similar to the product sequence of BSH. The level of expression of the bsh gene was altered in every concentration of bile salts in the growth medium with bsh genes from P. pentosaceus M103 were induced by bile salts at concentrations of 0.1% and 0.3% (w/v). We conclude that P. pentosaceus M103 may make suitable probiotic candidates that can tolerate conjugated bile acids.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.

Nisin Z Production by Wild Strains of Lactococcus lactis Isolated from Brazilian (Italian Type) Fermented Sausage

In this study, five bacteriocin-producing Lactococcus lactis strains were identified from different naturally fermented Brazilian sausages. Ion exchange and reversed-phase chromatographies were used to purify the bacteriocins from culture supernatant of the five strains. Mass spectrometry (MALDI-TOF/TOF) showed that the molecular masses of the bactericoins from L. lactis ID1.5, ID3.1, ID8.5, PD4.7, and PR3.1 were 3330.567 Da, 3330.514 Da, 3329.985 Da, 3329.561 Da, and 3329.591 Da, respectively. PCR product sequence analysis confirmed that the structural genes of bacteriocins produced by the five isolates are identical to the lantibiotic nisin Z. Optimal nisin Z production was achieved in tryptone and casein peptone, at pH 6.0 or 6.5. The most favorable temperatures for nisin Z production were 25°C and 30°C, and its production was better under aerobic than anaerobic condition. The type of carbon source appeared to be an important factor for nisin Z production. While sucrose was found to be the most efficient carbon source for nisin Z production by four L. lactis isolates, fructose was the best for one isolate. Lactose was also a good energy source for nisin Z production. Surprisingly, glucose was clearly the poorest carbon source for nisin Z production. The five isolates produced different amounts of the bacteriocin, L. lactis ID1.5 and ID8.5 isolates being the best nisin Z producers. DNA sequence analysis did not reveal any sequence differences in the nisZ and nisF promoter regions that could explain the differences in nisin Z production, suggesting that there should be other factors responsible for differential nisin Z production by the isolates.

A sensitive method for low input amount of exosomes in mouse model liquid biopsy using two-stage PCR

Background Recently, many potential non-invasive biomarkers have been developed using exosome-based liquid biopsy for early detection, diagnosis, and treatment of cancer. However, exosome analyses from small liquid biopsy samples have been limited with regard to sensitivity and efficiency. Methods We used a breast cancer model mouse with ERBB2 overexpression and collected liquid biopsy samples. We performed two-stage PCR for ERBB2, PPP1R1B, GRB7, and STARD3 genes. We compared conventional PCR methods with our two-stage PCR method. Both methods had same step of primer and cycle and amplification condition for RT-qPCR. PCR amplicons quality was validated by using the Sanger method. Results Quantitative analysis of all samples by conventional PCR compared with two-stage PCR template dilution (1/200) showed similar Ct values in all sample groups (cell, supernatant, urine, and plasma). Sequencing of the two-stage PCR-derived products revealed no changes in product sequence compared to that of conventional PCR. The obtained sequences showed 98-99% match of target gene sequences in the databases according to BLAST searches. Conclusion Two-stage PCR has better sensitivity and efficiency than conventional PCR methods for small-volume samples. Additionally, this method is useful for monitoring, as many genes can be assayed at once.

A Systematic Approach to Quality Oriented Product Sequencing for Multistage Manufacturing Systems

Product sequencing is one way to reduce cost and improve product quality for multistage manufacturing systems (MMS). However, systematically evaluating the influence of product sequence on quality performance for MMS is still a challenge. By considering the rate of incoming conforming product, manufacturing system quality transition between batch to batch, and quality propagation along stages, this paper investigates the appropriate batch policies and product sequencing for MMS so that satisfied quality performance can be achieved. A model to analyze the relationship between the product sequencing and quality performance is conducted just by using the quality inspection data and the complex engineering knowledge used in the variation method is avoided. Based on Markov Chain processes methodology, quality performance is modeled as a function of transition states jointly determined by multistage condition, product sequencing, incoming part quality, and propagation of the rate of conforming products among multistage. Quality related batch strategies are discussed for optimal quality performance. Two kinds of quality efficiency are put forward to facilitate the modeling and the discussion. The results of the model will lead to guidelines for quality management in multistage manufacturing systems.