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

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 (<i>e.g.</i>, 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 (<i>e.g.</i>, 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 <i>Escherichia coli</i>, producing colorful pigments. Moreover, C-Brick standard is also partially compatible with the BglBrick and BioBrick standards

CADS: CRISPR/Cas12a-Assisted DNA Steganography for Securing the Storage and Transfer of DNA-Encoded Information

Because DNA has the merit of high capacity and complexity, DNA steganography, which conceals DNA-encoded messages, is very promising in information storage. The classical DNA steganography method hides DNA with a “secret message” in a mount of junk DNA, and the message can be extracted by polymerase chain reaction (PCR) using specific primers (key), followed by DNA sequencing and sequence decoding. As leakage of the primer information may result in message insecurity, new methods are needed to better secure the DNA information. Here, we develop a pre-key by either mixing specific primers (real key) with nonspecific primers (fake key) or linking a real key with 3′-end redundant sequences. Then, the single-stranded DNA (ssDNA) <i>trans</i> cleavage activity of CRISPR/Cas12a is employed to cut a fake key or remove the 3′-end redundant sequences, generating a real key for further information extraction. Therefore, with the Cas12a-assisted DNA steganography method, both storage and transfer of DNA-encoding data can be better protected

MOESM2 of The draft genomes and investigation of serotype distribution, antimicrobial resistance of group B Streptococcus strains isolated from urine in Suzhou, China

Additional file 2: Figure S1. Positive results (iMLSB) in D-test. From left to right are No.10, No.11 and No.17, respectively

iBrick: A New Standard for Iterative Assembly of Biological Parts with Homing Endonucleases

<div><p>The BioBricks standard has made the construction of DNA modules easier, quicker and cheaper. So far, over 100 BioBricks assembly schemes have been developed and many of them, including the original standard of BBF RFC 10, are now widely used. However, because the restriction endonucleases employed by these standards usually recognize short DNA sequences that are widely spread among natural DNA sequences, and these recognition sites must be removed before the parts construction, there is much inconvenience in dealing with large-size DNA parts (<i>e.g</i>., more than couple kilobases in length) with the present standards. Here, we introduce a new standard, namely iBrick, which uses two homing endonucleases of I-SceI and PI-PspI. Because both enzymes recognize long DNA sequences (>18 bps), their sites are extremely rare in natural DNA sources, thus providing additional convenience, especially in handling large pieces of DNA fragments. Using the iBrick standard, the carotenoid biosynthetic cluster (>4 kb) was successfully assembled and the actinorhodin biosynthetic cluster (>20 kb) was easily cloned and heterologously expressed. In addition, a corresponding nomenclature system has been established for the iBrick standard.</p></div

Heterologous expression of the <i>act</i> cluster in <i>Streptomyces</i> 4F.

<p>(A) Constructed expression plasmid pIB2Am1_0-X000005. (B&C) Verification of pIB2Am1_0-X000005 with both the experimental digestion (B) and the electronic restriction (C) with XcmI. M, 1 kb DNA ladder with sizes labeled; -, pIB2Am1_0-X000004; +, pIB2Am1_0-X000005. For “−”, theoretical sizes are 8.32 and 1.88 kb; for “+”, sizes are 9.93, 5.28, 4.18, 3.38, 2.67, 2.08, 1.88, 1.08 and 0.78 kb. Same patterns were obtained for the experimental group and the electronic analysis group. (D) Heterologous expression of <i>act</i> cluster in <i>Streptomyces</i> 4F, employing pIB2Am1_0-X000004 as a negative control. Strains were cultured on R2YE plate at 30°C for 2 days.</p

Schematic diagram of iBrick forward assembly.

<p>(A) Construction of iBrick parts, which should be prefixed and suffixed in iBrick standard. (B) Forward assembly procedure. Part B is firstly released with I-SceI and PI-PspI digestion, and is then inserted into the PI-PspI digested vector, obtaining an assembled part AB. Alternatively, the vector can also be cut with I-SceI followed by insertion of part B to obtain part BA, which is designated the reverse assembly.</p

Ligation efficiency of iBrick assembly.

<p>* Clones on plates were evenly divided into 4 even sections with only one counted and the total number was then roughly calculated as four times of the number.</p><p>Ligation efficiency of iBrick assembly.</p

<i>Mtb</i> MazG is required for resistance to RNS shown <i>in vitro</i> and <i>ex vivo</i>.

<p>(<b>A</b>) <i>mazG</i>-null <i>Mtb</i> is susceptible to killing by acid nitrite. Exponential phase cultures (OD₆₀₀∼0.5) were suspended in 7H9-OADC pH4.5 with or without 2.5 mM NaNO₂ and treated for 20 hours. wt, wild-type <i>Mtb</i>; Δ<i>mazG</i>, <i>mazG</i>-null <i>Mtb</i>; compl, the complemented mutant. Data shown are mean ± S.E. of triplicates. * <i>p</i><0.05 <i>vs</i> wt. (<b>B</b>) Quantitative real-time PCR analyses of <i>mazG</i> and <i>dosR</i> from <i>Mtb</i> treated with DETA/NO or acid nitrite. All data were normalized to the levels of <i>sigA</i>. The <i>dosR</i> gene was used as a positive control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003814#ppat.1003814-Voskuil1" target="_blank">[70]</a>. Results are expressed as the changes in expression levels compared to untreated samples. Mean ± S.E of three independent repeats. <b>C to E</b>, Survival of wild-type <i>Mtb</i>, the <i>mazG</i>-null mutant and the complemented mutant in resting (<b>C</b>), activated (<b>D</b>) or NMMA treated (<b>E</b>) cells of murine macrophage cell line RAW264.7. Shown is one representative result of two independent experiments.</p

iBrick elements used in this study.

<p>iBrick elements used in this study.</p

Akupressur - Komplement till traditionell terapi vid postoperativt illamående och kräkningar

Postoperativt illamående och kräkningar, PONV, har sedan anestesins begynnelse vållat patienten stort lidande. Tillståndet kan förlänga den postoperativa vistelsen och är en av de vanligaste orsakerna till oplanerad inläggning av patienter i samband med dagkirurgi. Syftet med föreliggande arbete var att undersöka den förebyggande effekten av akupressur som ensam intervention eller i kombination med viss farmakologisk antiemetisk terapi vid postoperativt illamående och kräkningar hos vuxna patienter efter allmänkirurgi. En systematisk litteraturstudie genomfördes och tio vetenskapliga artiklar inkluderades och kvalitetsbedömdes. Sökning utfördes i databaserna PubMed, EBSCO HOST och Cochrane Library. Dessutom genomfördes manuell sökning. Resultatet visade att akupressur på en speciell triggerpunkt, P6, har en förebyggande effekt mot PONV. Ondansetron och akupressur är lika effektivt mot PONV, medan Droperidol verkar ha bättre effekt än akupressur. Slutsatsen var att akupressur har en plats som profylaktisk antiemetika för att förebygga PONV.Postoperative nausea and vomiting (PONV) has since the beginning of anesthesia caused the patient great suffering. It can prolong the post-operative stay and is one of the most common reasons for unplanned admittance of patients in connection to day surgery. The aim of this work was to study the preventive effect of acupressure as the lone intervention or in combination with certain pharmacological antiemetic therapies for post-operative nausea and vomiting in adult patients who have undergone general surgery. A systematic literature review was conducted and ten articles were included and each study subjected to a quality assessment. A PubMed, EBSCO HOST and Cochrane Library Database were conducted and a manual search of the literature references completed the search. The results showed that acupressure at the P6 meridian point has a preventive effect against PONV and that Ondansetron and acupressure are similarly effective against PONV, while Droperidal seems to have a better effect than acupressure. The conclusion was that acupressure can be used prophylactic to prevent PONV