“…This successfully eliminated nonspecific amplification resulting from interaction between primers and its inhibiting effect on single molecule amplification (Figure 2b), which in turn significantly decreased the total number of PCRs needed to obtain the minimal number of smPCR clones required for synthesis of error-free DNA. The sites for the C–A primer (as well as the random bar coding bases to be discussed later on) at the termini of the target molecules are incorporated by either an a priori PCR (16) or during the synthesis of the molecule as part of the target sequence. Figure 2.Primer, dimers and anticipation.…”
Section: Resultsmentioning
confidence: 99%
“…To facilitate the simple identification of rare smPCRs that despite the measures reported above were still not performed on single molecules, we integrated another feature in our procedure, previously proposed for other smPCR applications (16). We incorporated oligos with three random bases at both ends of the synthetic DNA constructs that are to be cloned, effectively bar-coding the molecules with a four-letter code at six positions (4 6 = 4096 tags) (Figure 4a).…”
The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.
“…This successfully eliminated nonspecific amplification resulting from interaction between primers and its inhibiting effect on single molecule amplification (Figure 2b), which in turn significantly decreased the total number of PCRs needed to obtain the minimal number of smPCR clones required for synthesis of error-free DNA. The sites for the C–A primer (as well as the random bar coding bases to be discussed later on) at the termini of the target molecules are incorporated by either an a priori PCR (16) or during the synthesis of the molecule as part of the target sequence. Figure 2.Primer, dimers and anticipation.…”
Section: Resultsmentioning
confidence: 99%
“…To facilitate the simple identification of rare smPCRs that despite the measures reported above were still not performed on single molecules, we integrated another feature in our procedure, previously proposed for other smPCR applications (16). We incorporated oligos with three random bases at both ends of the synthetic DNA constructs that are to be cloned, effectively bar-coding the molecules with a four-letter code at six positions (4 6 = 4096 tags) (Figure 4a).…”
The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.
“…GH7 CBH from Phanerochaete chrysosporium ( Pc Cel7C) has previously been genetically modified 17 . Pc Cel7C variants with single site-directed mutations were constructed on and near the catalytic tunnel using an in vitro expression system, and demonstrated that some mutants possessed higher activities than those of wild type (WT) toward phosphoric acid swollen cellulose (PASC) and 4-methylumbelliferyl β-D-cellobioside (MUC) 18 , 19 . However, the molecular mechanism for the effect of amino acid mutations on enzymatic activity has yet to be elucidated.…”
The glycoside hydrolase family 7 (GH7) member cellobiohydrolase (CBH) is a key enzyme that degrades crystalline cellulose, an important structural component of plant cell walls. As GH7 CBH is a major component in the enzyme mixture used to degrade biomass into fermentable glucose in biorefineries, enhancing its catalytic activity will significantly impact development in this field. GH7 CBH possesses a catalytic tunnel through which cellulose substrates are threaded and hydrolysed. Despite numerous studies dissecting this processive mechanism, the role of amino acid residues in the tunnel remains not fully understood. Herein, we examined the respective contributions of nine amino acid residues in the catalytic tunnel of GH7 CBH from Talaromyces cellulolyticus by substitution with alanine. As a result, N62A and K203A mutants were found to possess significantly higher cellulase activities than wild type. Molecular dynamics simulations showed that the N62 residue interacted strongly with the cellulose substrate, impeding threading, while the N62A mutant allowed cellulose to proceed more smoothly. Furthermore, the W63 residue was observed to facilitate twisting of the cellulose substrate in our simulations. This study helps elucidate cellulose threading and provides insight into biomass hydrolysis.
“…The well-controlled and flexible environment of the cell-free system offers several advantages over conventional in vivo technologies (Jermutus et al, 1998;Shimizu et al, 2001;Jewett et al, 2002). In brief, cell-free translation offers an attractive and convenient approach to produce properly folded proteins on a laboratory scale, incorporate unnatural or labeled amino acids, screen PCR fragment libraries in a high-throughput format, and ex-press pharmaceutical proteins (Noren et al, 1989;Hanes and Pluckthun, 1997;Kigawa et al, 1999;Nakano et al, 2000;Norais et al, 2001;Kiga et al, 2002;Guignard et al, 2002;Sawasaki et al, 2002;Takahashi et al, 2002;Jewett and Swartz, 2004b).…”
Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and UTP. Substrate replenishment of consumed amino acids, CTP, and UTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.
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