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.

The authors have nothing to disclose.

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 Level T vectors containing multiple gene expression cassettes). We recommend first listing all the gene expression cassette combinations required and then mapping the workflow from Level 0 to Level T in silico. During this process, users should consider the Level 1 &#34;Dummy&#34; parts available in the Plant MoClo kit that allow for the assembly of non-sequential Level 1 vectors in Level T (e.g., Level 1 position 1 and position 3 vectors can be assembled together with &#34;Dummy&#34; part Level 1 position 2), which can reduce the overall number of assembly reactions and cloning steps required15.
DNA synthesis is typically the simplest method for building new Level 0 parts. However, when cloning is required (e.g., from plasmids or genomic DNA), optimizing the design of the primers used for amplification is important for maximizing the efficiency of subsequent Level 0 vector assembly. The two most critical steps in primer design are: 1) checking that the correct overhangs are included and are in the appropriate orientation for the forward and reverse primers (Figure 1 and Table 1), and 2) ensuring that the length of the primer sequence that anneals to the template is sufficiently long (18&#8722;30 bp) and that the Tm value for this sequence (ideally 58&#8722;62&#176;C) is similar for the primer pair (Figure 1A). If a sequence requires domestication, several strategies are available. For short sequences (e.g., &#60;200 bp), a pair of long forward and reverse primers can be designed in which the 3&#8242; ends anneal to each other (i.e., an overlap of &#62;20 bp) and form a double stranded sequence following amplification. For longer sequences, separate fragments of the sequence can be amplified that remove illegal restriction sites and then assembled using a Golden Gate assembly approach (Figure 2). If assembly efficiency with PCR products is poor, individual fragments of a Level 0 sequence can be cloned into the Level 1 universal acceptor vector (pAGM1311), validated, and then assembled together into the appropriate Level 0 acceptor vector15. A 2:1 insert:acceptor vector molar ratio is recommended for efficient Golden Gate assembly. However, for assemblies of only 2-3 vectors (e.g., two Level 0 parts and a Level 1 acceptor vector), combining ~100 ng of each regularly typically results in successful assemblies. The efficiency of assemblies does tend to decrease as the number of vectors used per reaction increases, resulting in a reduction in total numbers of white colonies following transformation (Figure 3).
Prior to conjugation, validation of finalized Level T vectors by restriction digest and PCR is recommended. Conjugal DNA transfer is a well stablished technique for cyanobacterial strains, including those that are not naturally transformable41,45. Important steps in the conjugation protocol include: 1) careful handling of the helper E. coli strain following overnight growth (e.g., avoid vortexing)35, 2) taking care to completely remove traces of the antibiotics used to grow helper and cargo E. coli strains, 3) an appropriate incubation period for the mixture of cargo and helper strains and cyanobacteria (e.g., a longer incubation period was critical for S. elongatus UTEX 2973), and 4) the initial transfer period of the cell mixture on membranes in LB-BG11 agar plates lacking antibiotics for 24 h.
Isolated cyanobacterial colonies should develop on the membrane within two weeks for Synechocystis PCC 6803 and S. elongatus UTEX 2973, otherwise it is likely that conjugation has failed. Several modifications to the protocol could then be tested, including 1) using a cyanobacterial culture with a higher starting density (e.g., OD750 = 1.5&#8722;2); 2) increasing the incubation period before transfer to the membrane; and 3) extending the initial incubation period on the membrane from 24 h to 48 h (i.e., to allow more time for the expression of the antibiotic resistance gene on the transferred vector). If conjugation still fails, alternative methods such as electroporation could be tried53. Confirming a transgenic cyanobacterial strain is axenic is important prior to further experimentation. Finally, it is good practice to confirm the size of the heterologous vector in the transgenic cyanobacterial strain. The latter requires DNA extraction (section 5), transformation into E. coli and selection (section 2.1), and vector validation (section 2.4).
The outlined promoter characterization protocol uses small culture volumes (i.e., 2 mL) as a means of achieving a high throughput screening methodology. Larger volumes could be used depending on the photobioreactor space available, which would help to mitigate culture evaporation issues. If high throughput screening with small culture volumes is required, it is essential to have high humidity within the growth chamber to inhibit evaporation. Evaporation during a growth experiment can be detrimental to the accuracy and validity of sample measurements. Do check culture volumes during and after the experiment to confirm how much evaporation has occurred.
For plate reader measurements, it is important to measure cultures at low densities, ideally OD750 &#60; 1, to ensure the acquisition of reliable and reproducible growth and fluorescence data. A linear relationship between cell number and OD750 is observed only within a specific range54. To establish this range, we recommend performing a serial dilution (e.g., from OD750 = 0.1&#8722;1.0) using a known transformant where eYFP fluorescence has been confirmed. Plotting absolute fluorescence against normalized fluorescence (eYFP fluorescence/OD750) will help to identify the linear working range of culture densities. Several plate readers include a &#34;gain&#34; feature to modify the sensitivity of the fluorescence detector. In this case, the gain value should be set to an appropriate level before beginning the experiment and not changed between different experimental runs or the data will not be directly comparable.
Although the operation of different flow cytometers will vary between manufacturers, it is important to take a blank reading of the medium solution to facilitate the identification and gating of the target cyanobacterial population from any background signal in the medium (Figure 7A,B). Following this, subtraction of the fluorescence value of the negative control sample (e.g., a wild type strain) will help to remove native autofluorescence (Figure 7C,D). The photomultiplier tube (PMT) voltage parameter in a flow cytometer has a similar function to the gain in a plate reader, i.e., increasing or decreasing the sensitivity of the detector to the intensity of the fluorescence signal. As with the plate reader, PMT voltage should be set to an appropriate level before beginning the experiment55. Once set, the PMT voltage value should be maintained between different experimental runs or the data will not be directly comparable.

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><tbody><tr><td>5-bromo-4-chloro-3-indolyl-&amp;beta;-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&amp;prime;-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 &amp;mu;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) &amp;micro;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 &lt;em&gt;E. coli&lt;/em&gt;</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.

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 ...

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15.9K Views.  University of Edinburgh. 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.

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

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Genetics

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 ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Bioengineering

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 ...

Stable Gene Integration in Chlorella vulgaris Using Agrobacterium tumefaciens

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Author Spotlight: Optimized Transformation Protocol for Chlorella vulgaris Using Agrobacterium tumefaciens 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

Protocol for Co-Culturing Sucrose-Secreting Cyanobacteria with Fungal Partners

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

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

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Genetic Modification and Recombination of Salivary Gland Organ Cultures

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna