Name: genetic modification of cyanobacteria by conjugation using the cyanogate modular cloning toolkit
Uploaded: 2019-10-31T15:00:00
Description: Watch this Scientific Journal Video about Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit at JoVE.com

The authors are grateful to the PHYCONET Biotechnology and Biological Sciences Research Council (BBSRC) Network in Industrial Biotechnology and Bioenergy (NIBB) and the Industrial Biotechnology Innovation Centre (IBioIC) for financial support. GARG, AASO and AP acknowledge funding support from the BBSRC EASTBIO CASE PhD program (grant number BB/M010996/1), the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACYT) PhD program, and the IBioIC-BBSRC Collaborative Training Partnership (CTP) PhD program, respectively. We thank Conrad Mullineaux (Queen Mary University of London), and Poul Eric Jensen and Julie Annemarie Zita Zedler (University of Copenhagen) for plasmid vector and protocol contributions and advice.

1. Vector assembly using the Plant MoClo and CyanoGate toolkits
NOTE: Before proceeding with vector assembly, it is strongly recommended that users familiarize themselves with the vector level structures of the Plant and CyanoGate MoClo systems12,15.
<ol>
	<li>Construction of Level 0 parts 
	NOTE: Level 0 parts can be synthesized as complete vectors or as linear sequences for assembly with Level 0 acceptors (e.g., gBlocks, IDT)....

Golden Gate assembly has several advantages compared to other vector assembly methods, particularly in terms of scalability20,21. Nevertheless, setting up the Golden Gate system in a lab requires time to develop a familiarity with the various parts and acceptor vector libraries and overall assembly processes. Careful planning is often needed for more complex assemblies or when performing a large number of complex assemblies in parallel (e.g., making a suite of Le...

Cyanobacteria are autotrophic bacteria that can be used for the biosynthesis of a wide variety of natural and heterologous high value metabolic products1,2,3,4,5,6. Several hurdles still need to be overcome to expand their commercial viability, most notably, the relatively poor yields compared to heterotrophic bio-platforms (e.g., Escherichia coli and yeast)7. The recent expansion of available genetic engineering tools and uptake of the synthetic biology paradigm in cyanobacterial research is helping to overcome such challenges and further develop cyanobacteria as efficient biofactories8,9,10.
The main approaches for introducing DNA into cyanobacteria are transformation, conjugation and electroporation. The vectors transferred to cyanobacteria by transformation or electroporation are &#34;suicide&#34; vectors (i.e., integrative vectors that facilitate homologous recombination), while self-replicating vectors can be transferred to cyanobacteria by transformation, conjugation or electroporation. For the former, a protocol is available for engineering model species amenable to natural transformation11. More recently, a modular cloning (MoClo) toolkit for cyanobacteria called CyanoGate has been developed that employs a standardized Golden Gate vector assembly method for engineering using natural transformation, electroporation or conjugation12.
Golden Gate-type assembly techniques have become increasingly popular in recent years, and assembly standards and part libraries are now available for a variety of organisms13,14,15,16,17. Golden Gate uses type IIS restriction enzymes (e.g., BsaI, BpiI, BsmBI, BtgZI and AarI) and a suit of acceptors and unique overhangs to facilitate directional hierarchical assembly of multiple sequences in a &#34;one pot&#34; assembly reaction. Type IIS restriction enzymes recognize a unique asymmetric sequence and cut a defined distance from their recognition sites to generate a staggered, &#34;sticky end&#34; cut (typically a 4 nucleotide [NT] overhang), which can be subsequently exploited to drive ordered DNA assembly reactions15,18. This has facilitated the development of large libraries of modular Level 0 parts (e.g., promoters, open reading frames and terminators) defined by a common syntax, such as the PhytoBricks standard19. Level 0 parts can then be readily assembled into Level 1 expression cassettes, following which more complex higher order assemblies (e.g., multigene expression constructs) can be built in an acceptor vector of choice12,15. A key advantage of Golden Gate-type assembly techniques is their amenability to automation at high-throughput facilities, such as DNA foundries20,21, which can allow for the testing of complex experimental designs that cannot easily be achieved by manual labor.
CyanoGate builds on the established Plant MoClo system12,15. To incorporate a new part into CyanoGate, the part sequence must first be domesticated, i.e., &#34;illegal&#34; recognition sites for BsaI and BpiI must be removed. In the case of a part coding for an open reading frame (i.e., a coding sequence, CDS), recognition sites can be disrupted by generating synonymous mutations in the sequence (i.e., changing a codon to an alternative that encodes for the same amino acid residue). This can be achieved by a variety of approaches, ranging from DNA synthesis to polymerase chain reaction (PCR) amplification-based strategies such as Gibson assembly22. Depending on the expression host being used, care should be taken to avoid the introduction of rare codons that could inhibit the efficiency of translation23. Removing recognition sites in promoter and terminator sequences is typically a riskier endeavor, as modifications may affect function and the part might not perform as expected. For example, changes to putative transcription factor binding sites or the ribosome binding site within a promoter could alter strength and responsiveness to induction/repression. Likewise, modifications to key terminator structural features (e.g., the GC rich stem, loop and poly-U tail) may change termination efficiency and effect gene expression24,25. Although several online resources are available to predict the activity of promoter and terminator sequences, and inform whether a proposed mutation will impact performance26,27, these tools are often poor predictors of performance in cyanobacteria28,29,30.. As such, in vivo characterization of modified parts is still recommended to confirm activity. To assist with the cloning of recalcitrant sequences, CyanoGate includes a low copy cloning acceptor vector based on the BioBrick vector pSB4K512,16,31. Furthermore, a &#34;Design and Build&#34; portal is available through the Edinburgh Genome Foundry to help with vector design (dab.genomefoundry.org). Lastly, and most importantly, CyanoGate includes two Level T acceptor vector designs (equivalent to Level 2 acceptor vectors)15 for introducing DNA into cyanobacteria using suicide vectors, or broad host-range vectors capable of self-replication in several cyanobacterial species32,33,34.
Here we will focus on describing a protocol for generating Level T self-replicating vectors and the genetic modification of Synechocystis PCC 6803 and Synechococcus elongatus UTEX 2973 (Synechocystis PCC 6803 and S. elongatus UTEX 2973 hereafter) by conjugation (also known as tri-parental mating). Conjugal transfer of DNA between bacterial cells is a well described process and has been previously used for engineering cyanobacterial species, in particular those that are not naturally competent, such as S. elongatus UTEX 297335,36,37,38,39,40,41. In brief, cyanobacterial cultures are incubated with an E. coli strain carrying the vector to be transferred (the &#34;cargo&#34; vector) and vectors (either in the same E. coli strain or in additional strains) to enable conjugation (&#34;mobilizer&#34; and &#34;helper&#34; vectors). Four key conditions are required for conjugal transfer to occur: 1) direct contact between cells involved in DNA transfer, 2) the cargo vector must be compatible with the conjugation system (i.e., it must contain a suitable origin of transfer (oriT), also known as a bom (basis of mobility) site), 3) a DNA nicking protein (e.g., encoded by the mob gene) that nicks DNA at the oriT to initiate single-stranded transfer of the DNA into the cyanobacterium must be present and expressed from either the cargo or helper vectors, and 4) the transferred DNA must not be destroyed in the recipient cyanobacterium (i.e., must be resistant to degradation by, for example, restriction endonuclease activity)35,42. For the cargo vector to persist, the origin of replication must be compatible with the recipient cyanobacterium to allow for self-replication and proliferation into daughter cells post division. To aid with conditions 3 and 4, several helper vectors are available through Addgene and other commercial sources that encode for mob as well as several methylases to protect from native endonucleases in the host cyanobacterium43. In this protocol, conjugation was facilitated by an MC1061 E. coli strain carrying mobilizer and helper vectors pRK24 (www.addgeneorg/51950) and pRL528 (www.addgene.org/58495), respectively. Care must be taken when choosing the vectors to be used for conjugal transfer. For example, in the CyanoGate kit the self-replicating cargo vector pPMQAK1-T encodes for a Mob protein12. However, pSEVA421-T does not44, and as such, mob must be expressed from a suitable helper vector. The vectors used should also be appropriate to the target organism. For example, efficient conjugal transfer in Anabaena sp. PCC 7120 requires a helper vector that protects the mobilizer vector against digestion (e.g., pRL623, which encodes for the three methylases AvaiM, Eco47iiM and Ecot22iM)45,46.
In this protocol we further outline how to characterize the performance of parts (i.e., promoters) with a fluorescent marker using a plate reader or a flow cytometer. Flow cytometers are able to measure fluorescence on a single cell basis for a large population. Furthermore, flow cytometers allow users to &#34;gate&#34; the acquired data and remove background noise (e.g., from particulate matter in the culture or contamination). In contrast, plate readers acquire an aggregate fluorescence measurement of a given volume of culture, typically in several replicate wells. Key advantages of plate readers over cytometers include the lower cost, higher availability and typically no requirement for specialist software for downstream data analyses. The main drawbacks of plate readers are the relatively lower sensitivity compared to cytometers and potential issues with the optical density of measured cultures. For comparative analyses, plate reader samples must be normalised for each well (e.g., to a measurement of culture density, typically taken as the absorbance at the optical density at 750 nm [OD750]), which can lead to inaccuracies for samples that are too dense and/or not well mixed (e.g., when prone to aggregation or flocculation).
As an overview, here we demonstrate in detail the principles of generating Level 0 parts, followed by hierarchical assembly using the CyanoGate kit and cloning into a vector suitable for conjugal transfer. We then demonstrate the conjugal transfer process, selection of axenic transconjugant strains expressing a fluorescent marker, and subsequent acquisition of fluorescence data using a flow cytometer or a plate reader.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0404</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate (ATP) disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>Agar (microbiology tested)</td>
			<td>Sigma-Aldrich</td>
			<td>A1296-500g</td>
			<td>Used in 8.3.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioline</td>
			<td>BIO-41026</td>
			<td>Used in 6.</td>
		</tr>
		<tr>
			<td>Attune NxT Flow Cytometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>-</td>
			<td>Used in 4.3.1.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>BpiI (BbsI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER1011</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>BsaI (Eco31I)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ER0291</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>Carbenicillin disodium</td>
			<td>VWR International</td>
			<td>A1491.0005</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Corning Costar TC-Treated flat-bottom 24 well plates</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td>Used in 4.1.3.</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP231-100</td>
			<td>Used in 3.6.2.</td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>Qiagen</td>
			<td>69104</td>
			<td>DNA extraction kit. Used in 5.</td>
		</tr>
		<tr>
			<td>FLUOstar Omega Microplate reader</td>
			<td>BMG Labtech</td>
			<td>-</td>
			<td>Used in 4.2.2.</td>
		</tr>
		<tr>
			<td>GeneJET Plasmid Miniprep Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K0503</td>
			<td>Plasmid purification kit. Used in step 2.3.2.</td>
		</tr>
		<tr>
			<td>Glass beads (0.5 mm diameter)</td>
			<td>BioSpec Products</td>
			<td>11079105</td>
			<td>Used in 5.2.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td>10021083</td>
			<td>Used in 2.3.1, 3.6.2.</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10356553</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Kanamycin sulphate (Gibco)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11815-024</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Membrane filters (0.45 &#956;m)</td>
			<td>MF-Millipore</td>
			<td>HAWP02500</td>
			<td>Used in 3.3.7</td>
		</tr>
		<tr>
			<td>Microplates, 96-well, flat-bottom (Chimney Well) &#181;CLEAR</td>
			<td>Greiner Bio-One</td>
			<td>655096</td>
			<td>Used in 4.2.1.</td>
		</tr>
		<tr>
			<td>Monarch DNA Gel Extraction Kit</td>
			<td>New England Biolabs</td>
			<td>T1020S</td>
			<td>Used in 1.1.2.2.</td>
		</tr>
		<tr>
			<td>Monarch PCR DNA Cleanup Kit</td>
			<td>New England Biolabs</td>
			<td>T1030</td>
			<td>DNA purification kit. Used in 1.1.2.3.</td>
		</tr>
		<tr>
			<td>Multitron Pro incubator with LEDs</td>
			<td>Infors HT</td>
			<td>-</td>
			<td>Shaking incubator with white LED lights. Used in 4.1.4.</td>
		</tr>
		<tr>
			<td>MyTaq DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21108</td>
			<td>Used in 7.1.</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td>Used in 2.3.3.</td>
		</tr>
		<tr>
			<td>One Shot TOP10 chemically competent E. coli</td>
			<td>Thermo Fisher Scientific</td>
			<td>C404010</td>
			<td>Used in 2.1.1.</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS) solution (10X concentrate)</td>
			<td>VWR International</td>
			<td>K813</td>
			<td>Used in 4.3.2.</td>
		</tr>
		<tr>
			<td>Q5High-Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0491S</td>
			<td>Used in 1.1.2.1.</td>
		</tr>
		<tr>
			<td>Quick-Load 1 kb DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N0468S</td>
			<td>Used in Figure 4.</td>
		</tr>
		<tr>
			<td>Screw-cap tubes (1.5 ml)</td>
			<td>Starstedt</td>
			<td>72.692.210</td>
			<td>Used in 3.6.3</td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>VWR International</td>
			<td>J61820.06</td>
			<td>Used in 2.1.3.</td>
		</tr>
		<tr>
			<td>Sterilin Clear Microtiter round-bottom 96-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>612U96</td>
			<td>Used in 4.3.1.</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Thermo Fisher Scientific</td>
			<td>EL0011</td>
			<td>Used in Table 2.</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>Bead mill. Used in 5.2.</td>
		</tr>
	</tbody>
</table>

genetic modification of cyanobacteria by conjugation using the cyanogate modular cloning toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

To demonstrate the Golden Gate assembly workflow, an expression cassette was assembled in the Level 1 position 1 (Forward) acceptor vector (pICH47732) containing the following Level 0 parts: the promoter of the C-phycocyanin operon Pcpc560 (pC0.005), the coding sequence for eYFP (pC0.008) and the double terminator TrrnB (pC0.082)12. Following transformation of the assembly reaction, successful assemblies were identified using...

Watch this Scientific Journal Video about Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit at JoVE.com

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Cyanobacteria are an important phylum of photosynthetic microbes that are increasingly recognized as useful platforms for synthetic biology and renewable biotechnology applications. So developing robust techniques to simplify the design, cloning, and introduction of heterologous DNA into different cyanobacterial species is important. So this technique demonstrates a well-established method called conjugation or triparental mating for introducing DNA into cyanobacterial species that are not naturally transformable.Conjugation has been successfully performed in a wide array of cyanobacterial strains from single cell to multicellular filamentous species. If you are attempting this technique for the first time, please take care to handle both your cyanobacterial and your E.coli strains with care. For example, do not centrifuge above the values given in the protocol and do not vortex.Mixing bacteria and Cyanobacteria may seem strange to first-time users. We hope that by demonstrating the conjugation process, we will help to clarify this. After construction of level one assemblies, choose an appropriate level T acceptor vector.Choose an appropriate end link to ligate the three prime end of the final level one assembly to the level T backbone. To assemble one or more level one assemblies in level T, prepare a reaction mix with PBI1 and the required end link vector. Set up the thermal cycler program and proceed according to the manuscript.On day one, grow cyanobacterial culture by setting up a fresh culture of Synechocystis PCC 6803 or Synechococcus elongatus UTEX 2973. With a heat sterile loop, scrape the cells from the axenic BG-11 agar plate and place into a 100 milliliter conical flask filled with 50 milliliters of fresh BG-11 medium to inoculate. Place the flask into a shaking incubator with illumination to grow the Synechocystis PCC 6803 cell cultures at 30 degrees Celsius at 100 RPM.For Synechococcus elongatus UTEX 2973, grow the cell cultures at 40 degrees Celsius at 100 RPM. After one to two days, draw one milliliter of the cell culture and measure on a spectrophotometer. The absorbance at OD 750 reaches 0.5 to 1.5.On day two, inoculate an MC1061 E.coli helper strain containing vectors PRK24 and PRL528 in five milliliters of LB medium with ampicillin at a concentration of 100 micrograms per milliliter and chloramphenicol at a concentration of 25 micrograms per milliliter. Grow at 37 degrees Celsius overnight at 225 RPM in a shaking incubator. Then inoculate five milliliters of LB medium containing appropriate antibiotics with the E.coli culture carrying the level T cargo vector.Grow the culture at 37 degrees Celsius overnight at 225 RPM in a shaking incubator. On day three, centrifuge the helper and the cargo E.coli overnight cultures at 3, 000 g for 10 minutes at room temperature. Discard the supernatant without disturbing the cell pellet.Wash the pellets by adding five milliliters of fresh LB medium without antibiotics. Resuspend the pellet by gently pipetting up and down and centrifuge to remove the supernatant. Repeat this washing step three times to remove residual antibiotics from the overnight culture.After the last wash, resuspend the cell pellet in half the volume of LB medium of the initial culture volume. Then combine 450 microliters of the helper strain with 450 microliters of the cargo strain in a two milliliter tube and leave at room temperature. Next, prepare the cyanobacterial culture by centrifuging one milliliter of cyanobacterial culture at 1, 500 g for 10 minutes at room temperature.Then discard the supernatant carefully without disturbing the cell pellet. Wash the pellet four times by adding fresh BG-11 medium of the same initial volume. Add 900 microliters of the washed cyanobacterial culture to 900 microliters of the combined E.coli strains in a two milliliter tube.Mix the cultures by gently pipetting up and down. Incubate the mixture at room temperature for 30 minutes for Synechocystis PCC 6803 or two hours for Synechococcus elongatus UTEX 2973. Centrifuge the mixture at 1, 500 g for 10 minutes at room temperature.Remove 1.6 milliliters of the supernatant and resuspend the pellet in the remaining supernatant. Place one 0.45 micron membrane filter on an LB BG-11 agar plate without antibiotics. Carefully spread 200 microliters of the E.coli/cyanobacterial culture mix on the membrane with a sterile spreader or a sterile bended tip.Seal the plate with paraffin film and place the plate into an illuminated incubator for 24 hours. After 24 hours, carefully transfer the membrane using flame sterilized forceps to a fresh BG-11 agar plate containing appropriate antibiotics to select for the cargo vector. Seal the plate with paraffin film and incubate under the same conditions as previously until colonies appear.Colonies typically appear after 7-14 days for Synechocystis PCC 6803 and 3-7 days for Synechococcus elongatus UTEX 2973. To select conjugants, using a heat sterile inoculating rod, select at least two individual colonies from the membrane and streak onto a new BG-11 agar plate containing appropriate antibiotics. Then inoculate a streak of cyanobacterial culture into a 15 milliliter centrifuge tube containing five milliliters of LB medium and incubate at 37 degrees Celsius overnight at 225 RPM in a shaking incubator.The clear culture confirms the absence of E.coli contamination. Following a sufficient growth period of seven days, pick individual axenic colonies to set up liquid cultures for long-term cryo storage or subsequent experimentation. In this study, golden gate assembly workflow was demonstrated.Following transformation of the assembly reaction, successful assemblies were identified using standard blue white screening of E.coli colonies. The eYFP expression cassette in the level one vector and the end link one vector were then assembled into a level T acceptor vector to give the vector cpcBA-eYFP. The assembled cpcBA-eYFP vector was verified by restriction digestion.Successful conjugal transfer of cpcBA-eYFP or the pPMQAK1-T vector resulted in the growth of up to several hundred colonies on the membrane for Synechocystis PCC 6803 and Synechococcus elongatus UTEX 2973. As expected for the strong Pcpc560 promoter, the values for normalized eYFP fluorescence from the plate reader and eYFP fluorescence per cell from the flow cytometer were high compared to the negative control. With both the plate reader and the flow cytometer, fluorescence values were higher in Synechococcus elongatus UTEX 2973 than in Synechocystis PCC 6803.Ensure you incubate for the minimum time according to which strain you are conjugating.

genetic-modification-cyanobacteria-conjugation-using-cyanogate

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Cells, Genomes, and Evolution

SCIENTISTS IN ACTION

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...