protocols-for-c-brick-dna-standard-assembly-using-cpf1

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Chinese Academy of Sciences

Recently, it has been proved that methylprednisolone has inhibition effect on the proliferation of endogenous neural progenitor cells (NPCs) after spinal cord injury (SCI). Similar effect has also been found on NPCs cultured in vitro. However, the mechanism remains to be fully delineated. The purpose of this study is to investigate the potential molecular mechanism of this effect in NPCs cultured in vitro by gene expression profiling. Fetal mouse brain-derived NPCs were divided into 2 groups: NPCs incubated with methylprednisolone as a model of the methylprednisolone treatment after SCI, and without methylprednisolone as the control group. After the cell quantitative analysis and CCK-8 assay, the microarray analysis was carried out. Genes differentially expressed between NPCs treated with and without methylprednisolone were extracted. It was observed that the expression of 143 genes, including many members of distinct families, such as hypoxia inducible factors and neurotransmitter receptors, were significantly changed in response to the methylprednisolone treatment. Our results provide global molecular insights into the mechanisms of methylprednisolone-induced proliferation inhibition effect and suggest that EdnrB may play an important role in this effect.

Analysis of methylprednisolone-induced inhibition on the proliferation of neural progenitor cells in vitro by gene expression profiling.

The molecular basis of autosomal dominant microcephaly, a disorder associated with small head circumferences that results in variable mental retardation, is largely unknown. In the present study, we conducted a variation analysis of the DPP6 gene in patients with autosomal dominant microcephaly and variable mental retardation. The copy number variation analysis of DPP6 was performed on DNA samples from 22 patients with microcephaly using high-resolution, array-based genomic hybridization, and sequence analysis was performed to screen mutations in another 50 microcephalic patients. Two de novo deletions and one missense mutation in familial microcephalic patients were identified. The transfection of plasmids encoding green fluorescent protein-pLLU2G-shDPP6 fusion proteins in mouse brains revealed that the decreased expression of the DPP6 gene slightly reduced the weight of the mouse brains and resulted in mouse learning disabilities compared with their wild-type littermates. Our data indicate that the loss-of-function variations in DPP6 are associated with autosomal dominant microcephaly and mental retardation. DPP6 appears to play a major role in the regulation of proliferation and migration of neurons in neurogenesis, most likely by participating in neuronal electrical excitability, synaptic integration, and plasticity.

Loss-of-function variation in the DPP6 gene is associated with autosomal dominant microcephaly and mental retardation.

Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.

Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli.

Four new phenolic glycosides, named 2-ethylene-3,5,6-trimethyl-4-phenol-1-O-β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside (1), 3-methoxy-4-O-β-d-apiofuranosyl-(1→2)-β-d-glucopyranosylpropiophenone (2), 3-hydroxy-1-(4-O-β-d-glucopyranosyl-3-methoxyphenyl)propan-1-one (3) and 4-hydroxy-3,5-bis(3'-methyl-2-butenyl)benzoic acid-O-β-d-glucopyranoside (4), were isolated from the ethanol extract of Ficus tikoua, together with six known compounds: 3,4,5-trimethoxyphenol-1-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (5), 3,4,5-trimethoxyphenol-1-O-β-d-glucopyranoside (6), 3-methoxy-4-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranosylpropiophenone (7), baihuaqianhuoside (8), 3,5-dimethoxy-4-hydroxybenzoic acid-O-β-d-glucopyranoside (9) and 2-methoxy-4-allylphenyl-1-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (10). The structures of the four new compounds were elucidated by chemical methods and MS and IR, as well as 1D and 2D NMR analyses. The cytotoxicities of the 10 compounds against HeLa, K562, HL60 and HepG2 cell lines were assessed.

Phenolic glycosides from Ficus tikoua and their cytotoxic activities.

The radiation spectrum from the plasmas contains a large amount of information of plasmas. Thus, one of the most effective methods to detecting the plasma parameters is measure the plasma radiation spectrum. Until now, since the restriction of the Toshiba mechanically ruled aberration-corrected concave gratings, the measurable wavelength range of the incidence flat-field grazing spectrometer in the soft X-ray range are only from 5 to 40 nm. In order to extend the wavelength rang of grazing incidence flat-field spectrometer, first, a grazing incidence concave reflection grating ray-trace code is written using optical path equation. Second, under the same conditions with reference 6, we compare our numerical results with Harada's results. The results show that our results agree very well with the results of Harada. The results of comparison show that our ray-trace code is believable. Finally, the variety of the flat-field curves are detailedly investigated using the ray-trace code with the different grazing incidence conditions. The results show that the measurable wavelength range of the incidence flat-field grazing spectrometer are extended to 5~80 nm from the soft X-ray wavelength range of 5~40 nm. This result theoretically demonstrates the possibility of expanded the traditional band flat-field grazing incidence spectrometer from soft X-ray band to the extreme ultraviolet (XUV), and also bring a new design ideas for improving the use of grazing incidence flat field concave grating.

[A Concept Design of Flat-Field Spectrograph for Wide Wavelength Range].

So far, several DNA assembly standards have been developed, enabling scientists to conveniently share and modify characterized DNA parts. However, a majority of the restriction endonucleases used in these standards bear short recognition sites (e.g., 6 bps in BioBrick standard), which are widely distributed and need to be removed before further construction, causing much inconvenience. Although homing endonucleases, which recognize long DNA sequences, can be used for DNA assembly (e.g., iBrick standard), long scars will be left between parts, limiting their application. Here, we introduce a new DNA assembly standard, namely C-Brick, which employs the newly identified class 2 type V CRISPR-Cas systems protein Cpf1 endonuclease. C-Brick integrates both advantages of long recognition sites and short scars. With C-Brick standard, three chromoprotein cassettes were assembled and further expressed in Escherichia coli, producing colorful pigments. Moreover, C-Brick standard is also partially compatible with the BglBrick and BioBrick standards.

C-Brick: A New Standard for Assembly of Biological Parts Using Cpf1.

As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.

The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.

Accompanied with the internal non-homologous end joining (NHEJ) system, Cas9 can be used to easily inactivate a gene or delete a fragment through introduction of DNA double-stranded breaks (DSBs) in eukaryotic cells. While in most prokaryotes (e.g. Escherichia coli), due to the lack of NHEJ, homologous recombination (HR) is required for repair of DSBs, which is less convenient. Here, a markerless system was developed for rapid gene inactivation or fragment deletion in E. coli via introduction of both Cas9 and a bacterial NHEJ system. Three bacterial NHEJ systems, i.e. Mycobacterium smegmatis (Msm), Mycobacterium tuberculosis (Mtb) and Bacillus subtilis (Bs), were tested in E. coli, and the MsmNHEJ system showed the best efficiency. With the employment of Cas9 and MsmNHEJ, we efficiently mutated lacZ gene, deleted glnALG operon and two large DNA fragments (67 kb and 123 kb) in E. coli, respectively. Moreover, the system was further designed to allow for continuous inactivation of genes or deletion of DNA fragments in E. coli. We envision this system can be extended to other bacteria, especially those with low HR efficiency.

An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis.

Shi-Yuan Li

Institute of Plant Physiology and Ecology,
Shanghai Insititutes for Biological Sciences,
Institute of Plant Physiology and Ecology, Shanghai Insititutes for Biological Sciences

Chinese Academy of Sciences

Protocols for C-Brick DNA Standard Assembly Using Cpf1

Shi-Yuan Li

Institute of Plant Physiology and Ecology,Shanghai Insititutes for Biological Sciences,Institute of Plant Physiology and Ecology, Shanghai Insititutes for Biological Sciences

Chinese Academy of Sciences

Protocols for C-Brick DNA Standard Assembly Using Cpf1

Institute of Plant Physiology and Ecology,
Shanghai Insititutes for Biological Sciences,
Institute of Plant Physiology and Ecology, Shanghai Insititutes for Biological Sciences