Biology subjects

ma, x.

Publications and source records attributed to ma, x..

3 recordsLinked to original sources

bioRxiv2024

Cas9AEY (Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector) method of constructing large-sized DNA.

Saccharomyces cerevisiae is widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector can replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure plasmid. Saccharomyces cerevisiae cells are widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector has the ability to replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure knocked out plasmid.

synthetic biology↗

bioRxiv2024

A new method and application of PacBio sequencing for low copy and difficulty preparation plasmids.

The Single Molecule Real Time (SMRT) system developed by Pacific Biosciences applies the principle of synthesis while sequencing and uses the SMRT chip as the sequencing carrier. The high starting amount and good integrity of DNA required by PacBio library construction has always been a headache. Generally, the total loading volume of PacBio sequencing is 10g, and the concentration is not less than 200ng/L, which is a huge challenge for low-copy plasmids. Tight plasmid means that when the bacterial chromosome replicates once, the plasmid replicates once, and each bacterium only contains 1 to 2 plasmids. The replicon of the plasmid determines the copy number of the plasmid. Low copy plasmids should produce 0.2-1 g DNA per ml of LB culture. Low-copy plasmids play a key role in gene synthesis. When vectors are used for expression or other purposes, low-copy plasmids are used to reduce resource consumption caused by plasmid expansion. Low-copy plasmids are also used when plasmids have lethal gene clones. In order to solve the problem of low copy plasmid library database construction in PacBio, we used Phi29 polymerase to perform multiple substitution amplification of low copy plasmid, so as to obtain a large number of high molecular weight DNA to meet the computer requirements of PacBio. In addition, this study also established a PacBio sequencing method for bacterial fluids without the need for plasmid extraction steps, thereby reducing time and money costs.

molecular biology↗

bioRxiv2024

A method for filtering abnormal modified base calling in Oxford Nanopore Technologies sequencing

Backgroundgene synthesis sequencing using the long-read Oxford Nanopore Technologies (ONT) provides a cost-effective option for gene synthesis quality control. Despite the advantage of using long reads, however, accurate base calling is influenced by modified bases. ResultsWe introduce a method for filtering abnormal modified base calling in Oxford Nanopore Technologies sequencing. This method is based on the mapping results and perform an exact binomial test on the proportion of single base forward and reverse chain depth to determine the presence of abnormal modified base calling. Based on Sanger sequencing results, this method detected 96.79% of effective abnormal modified base calling, and the accuracy of gene synthesis sequencing increased from 99.40% before calibration to 99.98% after calibration, significantly reducing the proportion of requiring secondary gene synthesis sequencing. ConclusionThis method can accurately filter abnormally modified base calling sites, reduce the proportion of using Sanger for secondary sequencing, thereby saving costs, and improving efficiency.

bioinformatics↗