Abstract:Incorporation of synthetic degenerate oligonucleotides into plasmids for building highly diverse genetic libraries requires efficient and quantitative DNA manipulation. We present a fast and seamless method for generating libraries of PCR-synthesized plasmids designed with a degenerate sequence and short overlapping ends. Our method called QuickLib should find many applications in synthetic biology; as an example, we easily prepared genetic libraries of Escherichia coli expressing billions of different backbon… Show more
“…They are, however, in contrast to the report of Puspasari et al [36] who digested raw starch–degrading α-amylase from Bacillus aquimaris MKSC6.2 using Eco RI and Eco RV during a gene expression study. The choice of restriction enzyme to use when considering digestion of the partial amylase gene ( CDF_Amyl ) in molecular biology studies, however, will not only depend on the choice of restriction enzyme but also on a number of other factors such as the size of gene fragment required, the ability of the choice restriction enzyme to cut both the potential vector of interest and the amylase gene ( CDF_Amyl ), as well as the goal or nature of the research being considered, etc [18] , [29] , [43] . Fig.…”
In this investigation, a gene (CDF_Amyl) encoding extracellular α-amylase in Aspergillus niger strain CSA35 associated with cassava spoilage was amplified using specific primers and characterized in silico. The gene had a partial nucleotide sequence of 968 bp and encoded a protein of 222 aa residues with a molecular weight and isoelectric point of 25.13 kDa and 4.17, respectively. Its catalytic site was located in the active site domain. BLASTp analysis showed that the protein primary sequence of the α-amylase gene had 98% and 99% homologies with the α-amylase of A. niger and A. oryzae RIB40, respectively. The gene is more closely related to α-amylase genes from fungi than to bacterial, plant, or animal α-amylase genes. Restriction mapping of the gene showed it can be digested with restriction enzymes like NcoI, PstI, SmaI, and BcLI among others but not with EcoRI and EcoRV. Its protein product had a hydrophobicity score of − 0.43 but no transmembrane helix. The CDF_Amyl protein was subcellularly localized in the secretory pathway, an indication of its release into extracellular space after secretion. Also, the 3D structure of the CDF-Amyl protein was barrel-shaped with domains characteristic of α-amylases. The encoded α-amylase Vmax is 6.90 U/mg protein and Km is 6.70 mg/ml. It was concluded that the unique characteristics of the CDF_Amyl gene and its deduced protein could find applications in biotechnological, food and pharmaceutical industries where cloning and further modification of this gene would be required for product development and improvement.
“…They are, however, in contrast to the report of Puspasari et al [36] who digested raw starch–degrading α-amylase from Bacillus aquimaris MKSC6.2 using Eco RI and Eco RV during a gene expression study. The choice of restriction enzyme to use when considering digestion of the partial amylase gene ( CDF_Amyl ) in molecular biology studies, however, will not only depend on the choice of restriction enzyme but also on a number of other factors such as the size of gene fragment required, the ability of the choice restriction enzyme to cut both the potential vector of interest and the amylase gene ( CDF_Amyl ), as well as the goal or nature of the research being considered, etc [18] , [29] , [43] . Fig.…”
In this investigation, a gene (CDF_Amyl) encoding extracellular α-amylase in Aspergillus niger strain CSA35 associated with cassava spoilage was amplified using specific primers and characterized in silico. The gene had a partial nucleotide sequence of 968 bp and encoded a protein of 222 aa residues with a molecular weight and isoelectric point of 25.13 kDa and 4.17, respectively. Its catalytic site was located in the active site domain. BLASTp analysis showed that the protein primary sequence of the α-amylase gene had 98% and 99% homologies with the α-amylase of A. niger and A. oryzae RIB40, respectively. The gene is more closely related to α-amylase genes from fungi than to bacterial, plant, or animal α-amylase genes. Restriction mapping of the gene showed it can be digested with restriction enzymes like NcoI, PstI, SmaI, and BcLI among others but not with EcoRI and EcoRV. Its protein product had a hydrophobicity score of − 0.43 but no transmembrane helix. The CDF_Amyl protein was subcellularly localized in the secretory pathway, an indication of its release into extracellular space after secretion. Also, the 3D structure of the CDF-Amyl protein was barrel-shaped with domains characteristic of α-amylases. The encoded α-amylase Vmax is 6.90 U/mg protein and Km is 6.70 mg/ml. It was concluded that the unique characteristics of the CDF_Amyl gene and its deduced protein could find applications in biotechnological, food and pharmaceutical industries where cloning and further modification of this gene would be required for product development and improvement.
“…The first would involve performance of repeated randomizations and transformation positive colony collections until the desired number of colonies or the level of variations determined by NGS is reached, while the second would involve employment of entirely different circularization strategy. In our opinion the best alternative would be Gibson assembly, because several authors have reported acquisition of up to 10 8 of colony forming units [49]. An additional advantage of Gibson assembly would be the correction of possible mismatches between strands, because, due to close proximity to the end of DNA fragment, randomized region for one of the strands shall be destroyed and rebuilt anew using remaining strand as template.…”
Section: Discussionmentioning
confidence: 99%
“…Since such strategy is laborious, costly and low throughput, it usually resulted in acquisition of low data amounts, but nowadays with the widespread availability of massive parallel sequencing technologies (also known as NGS) this limitation is largely overcome [15,53,55,56]. However, surprisingly, we also observed that in a number of reports particularly in those that claimed development of "novel and highly effective" randomization strategy NGS data was not presented [49,57], which indicates that applicability of presented methodology to selected tasks should be reviewed critically and presented claims considered with caution.…”
mentioning
confidence: 82%
“…Consequentially, this bias problem is also acute in the case of single site randomization, because the same methods that are used for site and residue specific alterations are also employed here [23,47,49,50]. As one can see from information that was provided earlier, majority of these methods are PCR or other type of template based, thus also in here the if the target motif is larger than few codons then oligonucleotides with the greatest similarity to template shall bind/anneal with greater efficiency than others and the whole process shall result in acquisition of library with decreased sequence diversity.…”
Over the decades the improvement of naturally occurring proteins and creation of novel ones has been the primary goal for many practical biotechnology researchers and it is widely recognized that randomization of protein sequences coupled to various effect screening methodologies is one of the most powerful techniques for fast, efficient and purposeful approach for acquisition of desired improvements. Over the years considerable advancements have been made in this field, however development of PCR based or template guided methodologies has been hampered by the resulting template sequence bias. In this article we present novel whole plasmid amplification based approach, which we named OverFlap PCR, for randomization of virtually any region of the plasmid DNA, without introduction of mentioned bias.
“…Applications of Gibson assembly include site-directed mutagenesis and library construction [243]. A recent adaptation, QuickLib, is a modified Gibson assembly method that has been used to generate a cyclic peptide library [244]. QuickLib uses two primers that share complementary 5' ends; one long partially degenerate, and the other short non-degenerate, which are then used for full plasmid PCR amplification.…”
Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.
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