Genetic Constructor: An Online DNA Design Platform

Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community

Analysis of pro- and anti-viral cellular pathways.

<p>Left hand panels show selected fluorescence images of infected HeLa cells transfected with the indicated siRNAs at 48h post infection. Blue = DAPI (DNA stain), red = phalloidin (actin cytoskeleton) and green = VACV-A5eGFP. The z-score of each siRNA is indicated in the bottom right of each image. The right hand panels show the plot of sorted z-scores from the primary screen with the position of genes of interest marked. (a) Transcriptional proteins inhibitory for VACV replication (b) Anti-viral function of septins (c) Genome maintenance and DNA repair proteins inhibitory for VACV replication (d) The AMP-activated kinase complex is involved in VACV replication.</p

Validation of <i>Vaccinia virus</i> HFs.

<p>(a) Validation of primary screen hits using plaque assays. siRNA SMARTpools targeting five genes identified in the primary RNAi screen as modulating VACV growth (one anti-viral factor MAP3K14 and four pro-viral factors TRIP, PPAP2A, VPS52 and CCT7), and one non-specific SMARTpool (VP16) were transfected into HeLa cells and, after 48 h, infected at low MOI (0.05) with VACV-A5eGFP. At 12 h intervals, cells were collected and the amount of virus present calculated using a plaque assay. Results obtained in the primary RNAi screen are plotted on the right hand axis for comparison.</p

Identification of anti and pro-viral HFs common to multiple RNAi viral screens.

<p>Venn diagram showing the (a) pro-viral and (b) anti-viral hits common to at least two VACV RNAi screens and (c) hits common to the VACV screen reported in this study and three published influenza A RNAi screens with a total of 662 hits <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Brass1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Karlas1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Konig2" target="_blank">[47]</a> and three published HIV RNAi screens with a total of 826 hits <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Brass2" target="_blank">[41]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098431#pone.0098431-Zhou1" target="_blank">[43]</a>.</p

Identification of HFs for <i>Vaccinia virus</i> replication by RNA interference screen.

<p>(a) Schematic of the experimental workflow used to screen the replication of VACV with the druggable RNAi library. (b) Comparison of the level of fluorescence of the control siRNAs used in the primary screen. Wells were transfected with siRNA targeting PRK-AB1 and eGFP (known to downregulate VACV-A5eGFP growth), two negative controls (mock transfection and RSCF siRNA) and two non-specific siRNAs (targeting VP16 or VP11/12 from <i>Herpes simplex virus</i> type 1). Error bars indicate the standard error of the mean. (c) Correlation between level of fluorescence and amount of virus present. HeLa cells were mock transfected or transfected with siRNA which is not processed by the RISC machinery (RSCF) or which knocks down a strong VACV pro-viral factor (FBXL11). After 48 h cells were infected with VACV-A5eGFP at low multiplicity of infection (MOI 0.05). At 24, 36 and 48 h post infection fluorescence was measured (y axis) before the cells were collected for titration using a plaque assay (x axis). Correlation (Pearson product moment correlation coefficient) between the two datasets = 0.86. (d) Plot of sorted z-scores representing the level of fluorescence associated with each of the 6 719 siRNA SMARTpools in the screen (average of 8 replicates). siRNA pools targeting genes of particular interest are marked.</p

Transcriptional modulation of <i>Vaccinia virus</i> HFs.

<p>Plot of seven VACV HFs identified in the RNAi screen that are also strongly transcriptionally regulated in VACV infected cells. The x-axis represents the level of fluorescence in the RNAi screen (viral replication) expressed as a z-score with pro-viral genes to the left and anti-viral genes to the right. The y-axis represents the relative expression of the seven genes in VACV infected cells.</p

In situ activation of cellular genes TNFα, IFNβ and IL10 during infection of the liver.

<p>Immune compromised female BALB/c mice (n = 3 per group) were infected with 1×10⁵ PFU of MCMV or MCMVdie1 or not infected, i.e. uninfected but immune compromised mice to take into account that a 6.5 Gy total-body γ-irradiation by itself slightly stimulates the expression of the genes under investigation. The analysis was performed 10 days after infection. (A) Expression levels (ΔΔC_T values) relative to β-actin transcripts. (B, C) Normalisation of the relative expression levels of TNFα, IFNβ, and IL10 to the numbers of E1 transcripts per 500 ng of total RNA (B) or to the numbers of infected MCP⁺ cells per representative 10-mm² areas of liver tissue sections (C) in order to take account of differences in tissue infection density. Throughout, bars represent median values for three mice per experimental group. Variance bars indicate the range. P values for significance are indicated for group comparisons of interest (unpaired <i>t</i>-test, two-sided, performed with log-transformed data).</p

Growth of MCMV and MCMVdie1 and TNFα levels in different organs of infected BALB/c mice.

<p>Groups of BALB/c mice (4 mice per group) were inoculated with either 3×10⁵ or 3×10⁶ PFU of parental MCMV or MCMVdie1, respectively. On day 4 and 7 post infection mice were killed and spleens, livers, kidneys and hearts (A, B, C and D, respectively) were harvested, weighted, and sonicated as a 10% (wt/vol) tissue homogenates in DMEM. <b>Left panels.</b> Viral titres were determined by standard plaque assay on MEFs. Grey lines show limit of detection. Black horizontal marks show median values. <b>Right panels.</b> Levels of TNFα in different organs of infected BALB/c mice. At indicated times organs were harvested, weighted and homogenated as a 10% (wt/v). TNFα levels were determined by ELISA from the homogenates. Mock infected mice were also included as a negative control. No significant differences were found in production of infectious virus between infections, except in kidneys at 4 dpi. Statistically significant differences in TNFα levels are shown as * = p<0.05 or ** = p<0.01.</p

Cytokine production in infected MΦs.

<p>(A) Cells were mock-infected or infected with either MCMV or MCMVdie1 (MOI 1). IFNγ, IL10, IL12p70 and TNFα levels from cellular supernatants at 10 hpi were measured by flow cytometry-based CBA. (B) TNFα production after infection of RAW 264.7 macrophages. Cells were either mock-infected or infected with MCMV, MCMVdie1 or the MCMV IE1stop mutant (MOI 1). TNFα levels from the supernatants were determined by ELISA at 10 hpi. (C) TNFα production after infection of BMMΦ with MCMV, MCMVdie1 or MCMVdie3. Cytokine levels from cellular supernatants were measured by flow cytometry based CBA for 12 h time course, 16 and 24 hpi. (D) TNFα production from mock-infected BMMΦ or MCMV-, MCMVdie1- or MCMVrev-infected cells after 10 and 24 h. Cytokine levels were measured by ELISA. Experiments were done in triplicate and tested in duplicates. Bars show mean values with SE.</p

Investigation of IE1-mediated NF-κB, p38 or JNK activation.

<p>BMMΦs were mock infected or infected at an MOI of 1 with MCMV, MCMVdie1 or MCMVrev (n = 3). (A) Western blots for IκBα and the activated forms of the p38 and JNK kinases are shown. (B) Western Blots with transfected and infected RAW cells after LPS stimulation. RAW cells were transfected with IE1 expression plasmid pp89UC or a control vector (pEYFP-C2) and stimulated with 10 ng/ml LPS for 15 min or infected (MOI 1) and stimulated at 4 and 10 hpi, respectively.</p