The methodologies of the directed evolution benefit from quantitative reading quantitatively genotype at the phenotype. We have therefore designed a method flowing from protein-peptide interactions to dynamic reading provided by a modified polymerase DNA. The melting of a processivity clamping protein on a polymerase thermostable nucleic acid allows a polymerase activity and an amplification of the DNA in high prohibition salt buffers. Here, we recap this phenotype by indirectly coupling the SSO7D processing clamp to the TAQ DNA polymerase via a respective fusion to a pair of high affinity peptide proteins and thermostable interaction. Escherichia coli cells co-expressing peptide-peptide-peptide pairs can be used directly in polymerase chain reactions to determine the relative interaction resistors by measuring amplic yields. The conditional activity of the polymerase is also used to connect the genotype to the phenotype of the interactive peptide pairs co-expressed in E. coli using the phased evolution platform for the compartmentalized automatic replication.
We validate this approach, called the replication with two hybrid compartmentalized, by selecting for the high affinity peptides that bind two models of model proteins: Spycacher and the large fragment of Nanoluc Luciferase. We demonstrate more co-evolution directed by hiking both protein and peptide components of the SpycCatcher-Spytag pair and by co-selecting for functional interactive variants. An important toxin-antitoxin (TA) HOK / Sok system, encoded by the plasmid R1 of Escherichia coli, is involved in the wake of post-segregation cells that have lost the plasmid. The deadly properties of HOK protein have been used for environmental containment of microbes and the development of potential vaccine candidates. We aim to demonstrate the powerful antimicrobial property of 19 fragment of the HOK peptide amino acid (AA) terminal.
This has been accomplished by designing a conditional suicide system based on HOK gene expression, cloned in an anhydroteracyclacin (ATC) inducible vector – PASK75. Thermal shocks and electroporation were used for the transformation of Escherichia coli cells and Vibrio Cholerae respectively. The minimum induction concentration (medium C) of the ATC, determined by analyzing the expression of the green fluorescent protein cloned separately in the PASK75 vector, was 30 ng / ml. As the HOK gene has been synthesized by Novo (using the reaction of the recombinant polymerase chain) in our study, various random size HOK fragments were generated (as a result of the possible nature of Taq. polymerase).
Building better DNA polymerases: Engineering Replication of Expanded Genetic Alphabets
DNA polymerases are now used in all scientific research, biotechnology and medicine, in part for their ability to interact with non-natural forms of DNA created by synthetic biologists. Here, especially, natural DNA polymerases often do not have “performance specifications” needed for processing technologies. This creates a need for rational (or semi-rational) scientific engineering engineering in order to identify variants that reproduce non-natural basic pairs (UBP), non-natural backbones, tags or other features evolutionually new from the Non-natural DNA. In this review, we provide a brief overview of the chemistry and properties of the replica DNA polymerases and their evolved variants, with a focus on the Klenow fragment of Taq DNA polymerase (Klentaq).
We describe studies of structural, enzymatic and comparative molecular dynamics of wild-type variants and klentaq, complex with natural or non-canonic substrates. The combination of these methods makes it possible to understand how the specific amino acid substitutions remote from the active site in a variant of the Klentaq DNA polymerase (ZP Klentaq) contribute to its ability to reproduce non-natural basic pairs (UBP). With improved efficiency compared to Klentaq. This approach can therefore be used to guide any future rational engineering of replicative DNA polymerases.
Directed co-evolution of interacting protein-peptide pairs by compartmentalized two-hybrid replication (C2HR)
Evaluation of the comparability of different methods of DNA extraction and amplification in microbial community profiling of intestines
Automated and broadband technologies are becoming increasingly common in microbiome studies to reduce costs and increase efficiency. However, in microbiome studies, small differences in methodology – including storage conditions, wet laboratory methods, sequencing platforms and data analysis – can affect the reproducibility and comparability of data between the studies.
The effects of broadband methods have been limited, including microfluidic PCR technologies. In this article, we compare two extraction methods (the mini Qiaamp DNA stool kit and the Mobio PowerSoil DNA insulation kit), two Taq polymerase enzymes (Mytaq HS Red Mix and Lock II PCR Tougymix) , two sets of primers (V3-V4 and V4 -V5) and two amplification methods (a two-step PCR protocol and a preparation of the amplicon library on the fluidigm access network system that allows multiplexing automated primers). The profiles of the Gut microbial community have been considerably affected by all variables. Although there has been no significant difference in alpha diversity measured between the two extraction methods, there was an effect of the extraction method on the community composition measured by unrestricted unifruc distances. The amplification method and primers have had a significant effect on alpha diversity and community composition.
Description: DNA polymerase eta (POLH), is a protein that in humans is encoded by the POLH gene. This gene encodes a member of the Y family of specialized DNA polymerases. It copies undamaged DNA with a lower fidelity than other DNA-directed polymerases. However, it accurately replicates UV-damaged DNA; when thymine dimers are present, this polymerase inserts the complementary nucleotides in the newly synthesized DNA, thereby bypassing the lesion and suppressing the mutagenic effect of UV-induced DNA damage. This polymerase is thought to be involved in hypermutation during immunoglobulin class switch recombination. Mutations in this gene result in XPV, a variant type of xeroderma pigmentosum. Several transcript variants encoding different isoforms have been found for this gene.
Description: DNA polymerase iota is an enzyme that in humans is encoded by the POLI gene. The protein encoded by this gene is an error-prone DNA polymerase involved in DNA repair. The encoded protein promotes DNA synthesis across lesions in the template DNA, which other polymerases cannot do. The encoded polymerase inserts deoxynucleotides across lesions and then relies on DNA polymerase zeta to extend the nascent DNA strand to bypass the lesion.
Description: Polymerase (DNA directed), beta, also known as POLB, is an enzyme that, in humans, is encoded by the POLB gene. It is localized on 8p11.2. The protein encoded by this gene is a DNA polymerase involved in base excision and repair, also called gap-filling DNA synthesis. It is found that a truncated POLB is expressed in primary colorectal tumors and inhibits the normal repair function of wildtype POLB. The encoded protein, acting as a monomer, is normally found in the cytoplasm, but it translocates to the nucleus upon DNA damage. Several transcript variants of this gene exist, but the full-length nature of only one has been described to date. Additionally, human POLB forms a complex with and is methylated by PRMT6. In vitro, methylated POLB possesses significantly higher DNA polymerase activity when compared to that of unmodified enzyme. The increase in DNA polymerase activity upon methylation is due to the enhanced DNA binding and processivity of POLB.
Description: The protein encoded by this gene is a DNA polymerase involved in base excision and repair, also called gap-filling DNA synthesis. The encoded protein, acting as a monomer, is normally found in the cytoplasm, but it translocates to the nucleus upon DNA damage. [RefSeq]
Description: DNA polymerase lambda, also known as POLL, is a protein found in eukaryotes. In humans, it is encoded by the POLLA gene. POLL has 5-prime-deoxyribose-5-phosphate lyase activity and strand-displacement synthesis activity on gapped DNA substrates, suggesting that POLL participates in short- and long-patch base excision repair. POLL is widely expressed, with abundant expression in pachytene spermatocytes of testis and in ovary.
The relative abundance of actinobacteria was significantly lowered when using the Mobio Kit amplification method or fluidigm, and the relative abundance of firmoles was lower when using the Qiagen Kit. Microbial community profiles based on fluidigm-generated amplicon libraries have not been comparable to those generated by more commonly used methods. Researchers should carefully consider limitations and biases that different methods of extraction and amplification can introduce their results. In addition, a deeper benchmarking of automated methods and multiplexing is required to determine the magnitude of the potential compromise between the quality and the amount of data.
Jordan
