Additional file 1: Figure S1. of Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

Fluorescence images of CHO cells transfected with the plasma membrane labelled Lyn-Ceru and ER labelled STIM1-mRFP markers. CHO cells transfected with the plasma membrane labelled Lyn-Ceru showed a matte-like appearance (a, f and k) while those transfected with the ER labelled STIM1-mRFP showed a web-like fluorescence distribution (d, i and n). TM-Venus (b), TLP-Venus (g) or TLP-V-TM (l) peptides show a shift in fluorescence distribution from a wholly ER (b) to a slight (g) and then an entirely membrane appearance (l). Merged images illustrate resultant co-localization (c, e, h, j, m and o). TM: transmembrane domain TLR4, TLP: fusion of TM with signal peptidase cleavage site from human immunoglobulin K, V: Venus fluorescent protein, LC: Lyn-Ceru, STIM1: stromal interaction molecule 1. Scale bars are 10 Οm. Images are false colored: CFP, cyan; YFP, green; mRFP, red. All insets show zoomed regions (4x) of structures in dotted rectangles. All experiments were repeated at least 3 times. (PDF 9198 kb

Autonomous Cell Migration to CSF1 Sources <i>via</i> a Synthetic Protein-Based System

Inflammatory lesions, often seen in diseases such as rheumatoid arthritis, atherosclerosis and cancer, feature an acidic (<i>i.e.</i>, low pH) microenvironment rampant with cytokines, such as CSF1. For potential therapeutic intervention targeted at these CSF1 sources, we have assembled a system of four proteins inside a cell (<i>i.e.</i>, HEK293) that initially had no natural CSF1-seeking ability. This system included a newly engineered CSF1 chimera receptor (named CSF1Rchi), the previously engineered Ca²⁺ activated RhoA (<i>i.e.</i>, CaRQ), vesicular stomatitis virus glycoprotein G (VSVG) and thymidine kinase (TK). The binding of CSF1 to the CSF1Rchi generated a Ca²⁺ signal that activated CaRQ-mediated cellular blebbing, allowing autonomous cell migration toward the CSF1 source. Next, the VSVG protein allowed these engineered cells to fuse with the CSF1 source cells, upon low pH induction. Finally, these cells underwent death postganciclovir treatment, <i>via</i> the TK suicide mechanism. Hence, this protein system could potentially serve as the basis of engineering a cell to target inflammatory lesions in diseases featuring a microenvironment with high levels of CSF1 and low pH

Antibody-Based Fusion Proteins Allow Ca²⁺ Rewiring to Most Extracellular Ligands

The Ca²⁺ signaling toolkit is the set of proteins used by living systems to generate and respond to Ca²⁺ signals. The selective expression of these proteins in particular tissues, cell types and subcellular locations allows the Ca²⁺ signal to regulate a diverse set of cellular processes. Through synthetic biology, the Ca²⁺ signaling toolkit can be expanded beyond the natural repertoire to potentially allow a non-natural ligand to control downstream cellular processes. To realize this potential, we exploited the ability of an antibody to bind its antigen exclusively in combination with the ability of the cytoplasmic domain of vascular endothelial growth factor receptor 2 (VEGFR2) to generate a Ca²⁺ signal upon oligomerization. Using protein fusions between antibody variants (<i>i.e.</i>, nanobody, single-chain antibody and the monoclonal antibody) and the VEGFR2 cytoplasmic domain, Ca²⁺ signals were generated in response to extracellular stimulation with green fluorescent protein, mCherry, tumor necrosis factor alpha and soluble CD14. The Ca²⁺ signal generation by the stimulus did not require a stringent transition from monomer to oligomer state, but instead only required an increase in the oligomeric state. The Ca²⁺ signal generated by these classes of antibody-based fusion proteins can be rewired with a Ca²⁺ indicator or with an engineered Ca²⁺ activated RhoA to allow for antigen screening or migration to most extracellular ligands, respectively

Genetically Encoded Circuit for Remote Regulation of Cell Migration by Magnetic Fields

Magnetoreception can be generally defined as the ability to transduce the effects of a magnetic field into a cellular response. Magnetic stimulation at the cellular level is particularly attractive due to its ability for deep penetration and minimal invasiveness, allowing remote regulation of engineered biological processes. Previously, a magnetic-responsive genetic circuit was engineered using the transient receptor potential vanilloid 1 (TRPV1) and the iron containing ferritin protein (<i>i.e.</i>, the TF circuit). In this study, we combined the TF circuit with a Ca²⁺ activated RhoA protein (CaRQ) to allow a magnetic field to remotely regulate cell migration. Cells expressing the TF circuit and CaRQ exhibited consistent dynamic protrusions, leading to migration along a porous membrane, directed spreading in response to a magnetic field gradient, as well as wound healing. This work offers a compelling interface for programmable electrical devices to control the migration of living systems for potential applications in cell-based therapy

Additional file 2: Figure S2. of Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells

Line graph showing changes in distribution of plasma membrane labelled Lyn-Ceru compared with ER-labelled STIM1-mRFP, TM-Venus and TLP-Venus in CHO cells, post cyclohexamide treatment. CHO cells transfected with the plasma membrane labelled Lyn-Ceru and the ER labelled STIM1-mRFP, TM-Venus or TLP-Venus. Cells were imaged initially and after 1, 4 and 12 h after incubation with 10 μg/mL cyclohexamide. Y-axis shows PCC of the STIM1-mRFP, TM-Venus or TLP-Venus with Lyn-Ceru. TM: transmembrane domain TLR4, TLP: fusion of TM with signal peptidase cleavage site from human immunoglobulin K, V: Venus fluorescent protein, LC: Lyn-Ceru, STIM1: stromal interaction molecule 1. Error bars indicate s.d. Star indicates significance of p < 0.05. (PDF 238 kb

Engineering Synthetic Proteins to Generate Ca²⁺ Signals in Mammalian Cells

The versatility of Ca²⁺ signals allows it to regulate diverse cellular processes such as migration, apoptosis, motility and exocytosis. In some receptors (<i>e.g.</i>, VEGFR2), Ca²⁺ signals are generated upon binding their ligand(s) (<i>e.g.</i>, VEGF-A). Here, we employed a design strategy to engineer proteins that generate a Ca²⁺ signal upon binding various extracellular stimuli by creating fusions of protein domains that oligomerize to the transmembrane domain and the cytoplasmic tail of the VEGFR2. To test the strategy, we created chimeric proteins that generate Ca²⁺ signals upon stimulation with various extracellular stimuli (<i>e.g.</i>, rapamycin, EDTA or extracellular free Ca²⁺). By coupling these chimeric proteins that generate Ca²⁺ signals with proteins that respond to Ca²⁺ signals, we rewired, for example, dynamic cellular blebbing to increases in extracellular free Ca²⁺. Thus, using this design strategy, it is possible to engineer proteins to generate a Ca²⁺ signal to rewire a wide range of extracellular stimuli to a wide range of Ca²⁺-activated processes

Parts-Based Assembly of Synthetic Transmembrane Proteins in Mammalian Cells

Transmembrane proteins span cellular membranes such as the plasma membrane and endoplasmic reticulum (ER) membrane to mediate inter- and intracellular interactions. An N-terminal signal peptide and transmembrane helices facilitate recruitment to the ER and integration into the membrane, respectively. Using a parts-based assembly approach in this study, we confirm that the minimum requirement to create a transmembrane protein is indeed only a transmembrane helix (TM). When transfected in mammalian cells, our fusion proteins in the schematic form X-TM-Y were localized to vesicles, the golgi apparatus, the nuclear envelope, or the endoplasmic reticulum, consistent with ER targeting. Further studies to determine orientation showed that X was facing the cytoplasm, and Y the lumen. Lastly, in our fusion proteins with an N-terminal TM, the TM effectively reversed the orientation of X and Y. This knowledge can be applied to the parts-based engineering of synthetic transmembrane proteins with varied functions and biological applications

Parts-Based Assembly of Synthetic Transmembrane Proteins in Mammalian Cells

Transmembrane proteins span cellular membranes such as the plasma membrane and endoplasmic reticulum (ER) membrane to mediate inter- and intracellular interactions. An N-terminal signal peptide and transmembrane helices facilitate recruitment to the ER and integration into the membrane, respectively. Using a parts-based assembly approach in this study, we confirm that the minimum requirement to create a transmembrane protein is indeed only a transmembrane helix (TM). When transfected in mammalian cells, our fusion proteins in the schematic form X-TM-Y were localized to vesicles, the golgi apparatus, the nuclear envelope, or the endoplasmic reticulum, consistent with ER targeting. Further studies to determine orientation showed that X was facing the cytoplasm, and Y the lumen. Lastly, in our fusion proteins with an N-terminal TM, the TM effectively reversed the orientation of X and Y. This knowledge can be applied to the parts-based engineering of synthetic transmembrane proteins with varied functions and biological applications

Photoactivatable intein has spatial precision.

<p>(A) HeLa cells co-expressing RhoA_N-InN-mRFP and LOVInC-RhoA_C-Venus and viewed under low magnification (20x objective). Two groups of cells that have been identified by white arrows and shown enlarged in panels B and C. Cells located near the center of the field were photostimulated with periodic interval of blue-light (1 second every 0.5 min). (D) Low magnification of the same set of cells after 150 mins of showing cells within the illumination zone (dotted white circle) have undergone dynamic blebbing while cells outside the illumination zone generally remained unchanged. Again, the two groups of cells are shown enlarged in panels E and F. Scale bars are 50 μm for A and D and 30 μm for B, C, E, and F. Images are in false colour.</p

160-fold acceleration of the Smith-Waterman algorithm using a field programmable gate array (FPGA)-4

<p><b>Copyright information:</b></p><p>Taken from "160-fold acceleration of the Smith-Waterman algorithm using a field programmable gate array (FPGA)"</p><p>http://www.biomedcentral.com/1471-2105/8/185</p><p>BMC Bioinformatics 2007;8():185-185.</p><p>Published online 7 Jun 2007</p><p>PMCID:PMC1896180.</p><p></p>d by the following vector - (we = write enable for SRAM blocks, rm = reset 64×SCM matrix, ena_seq = enable sequences to be loaded, ena_sf = enable scores and flags to be loaded). To clear all scores and flags from the matrix, the FSM is set to the 'Reset' state. Next, the FSM remains in the 'Wait for Sequence Load' state until two sequences of length 8 or less have been loaded by the C program. Once this loading is completed, the C program will assert the done_load signal. At this point, the FSM releases the matrix's reset signal which causes the sequences, scores and flags to propagate through the matrix. After a set delay determined by the critical path of the circuit, the FSM asserts the done_sw signal, and enables the values just calculated to be written into the RAM. Theses scores and flags will be read from the RAM for the next block. The FSM then returns to the 'Wait for Sequence Load' state, and waits for the next length of sequences to come from the C program. This loop is repeated until the entire Smith-Waterman matrix has been calculated and the score of the optimal alignment has been determined. Finally, the results are printed to a command window on the computer. The FSM can be reset by writing to a status register, allowing the matrix to be used for another set of sequences