The permission by Springer and Elsevier to reprint figures is greatly acknowledged.

1. Isolation of E. coli Co-transformants
<ol>
	<li>Prepare electro-competent cells of the appropriate E. coli strain to be transformed. Transfer to 1 ml of LB medium (Tryptone, Yeast Extract, NaCl at 10, 5, and 10 g/L, respectively) a single colony of the strain of choice, and incubate at 37 &#176;C under shaking conditions of 180 rpm. Dilute the pre-culture 1:500 in 25 ml of fresh LB medium and incubate the culture at 37 &#176;C.</li>
	<li>At log-phase (0.6 O.D.) centrifuge the cells suspension at 5,000 x g for 20 min), and resuspend the pellet in 10%, v/v ice-cold sterile glycerol-water in half of the original culture volume. Repeat this step 4 times, halving each time the resuspension volume. Finally, resuspend the pellet in glycerol/water and divide the cells suspension in 50 &#956;l aliquots. Store the aliquots at -80 &#176;C up to 6 months.</li>
	<li>Dissolve the desired plasmid in sterile water supplemented with 0.5 mM EDTA. Thaw on ice an aliquot of electro-competent cells and mix with an appropriate amount (2.5-5 ng) of vector. Dispense the mixture into a 0.1 cm cuvette suitable for electroporation, and apply 1.8 kV.</li>
	<li>Immediately transfer the electroporated cells into 1 ml of SOC medium (LB medium supplemented with 0.2% w/v glucose, 10 mM MgCl2, 2.5 mM KCl), incubate for 1 hr under shaking, and finally transfer 100 &#956;l aliquots to Petri dishes containing LB agar with the appropriate antibiotic. Incubate O/N at 37 &#176;C.</li>
	<li>Purify the transformants by streaking single colonies on Petri dishes.</li>
	<li>Prepare electro-competent cells of the primary transformants and repeat steps 1.1 to 1.5 to transform with further plasmids.</li>
	<li>Prepare glycerol stocks of the co-transformants. Transfer a single colony in LB supplemented with the appropriate antibiotics, incubate at 37 &#176;C under shaking, and at log-phase (0.6 O.D.) centrifuge the cells suspension at 5,000 x g for 20 min. Resuspend the pellet in LB-antibiotics medium containing 15% v/v glycerol. Dispense in aliquots and store at &#8211; 80 &#176;C.</li>
</ol>
2. Co-expression of &#945; and &#949; Subunits of E. coli DNA Polymerase III
<ol>
	<li>Transfer with a sterile loop a small amount of the appropriate strain glycerol stock (e.g., TOP10/pGOOD-&#949;243 and TOP10/pGOOD-&#949;243-&#952;/pBAD&#945;1160, see Figure 1) into a Petri dish containing LB medium and antibiotics. Let the cells suspension dry onto the Petri dish, and streak the cells droplet. Incubate at 37 &#176;C O/N.</li>
	<li>Transfer, using a sterile toothpick, a single colony to 1 ml of LB-antibiotics medium. Incubate 8 hr at 37 &#176;C. Dilute the pre-culture 1:500 in fresh medium and incubate at 30 &#176;C for 15 hr.</li>
	<li>Add IPTG, arabinose, or arabinose and IPTG, 1 mM each. Incubate at 30 &#176;C for 2.5 hr. Collect the cells, and store the pellets at -20 &#176;C.</li>
	<li>Thaw pellets on ice and resuspend in lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 2.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8). Gently homogenize the cells suspension with a cold glass potter.</li>
	<li>Sonicate at 15 W for 15 sec, followed by 15 sec cooling interval (4 cycles).</li>
	<li>Centrifuge the total protein extracts at 10,000 x g for 20 min, at 4 &#176;C to recover the soluble fraction.</li>
	<li>Analyze by SDS-PAGE (12.5% acrylamide) aliquots of each soluble protein extract. To this aim, transfer 20 &#956;l of each soluble protein extract to Eppendorf tubes containing 60 &#956;l of H2O and 20 &#956;l of loading buffer (250 mM Tris-HCl pH 6.8, 10 mM &#946;-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol, 0.25% (w/v) bromophenol blue) and boil for 5 min. Load 18 &#956;l of each sample and perform the electrophoresis at 140 V for about 1.5 hr.</li>
</ol>
3. Co-expression of &#945; Subunit of Deinococcus radiodurans DNA Polymerase III and &#949; Subunit of E. coli DNA Polymerase III
<ol>
	<li>Prepare a pre-culture of the E. coli strain TOP10/pBAD-&#945;Dr/pGOOD-&#949;243 in 1 ml of LB-ampicillin-tetracycline medium and incubate 8 hr at 37 &#176;C. Dilute 1:1,000 in fresh medium and incubate O/N at 37 &#176;C.</li>
	<li>Dilute 1:100 into 200 ml of fresh medium, incubate at 37 &#176;C for 3 hr, then induce for 3 hr with arabinose and IPTG, 1 mM each. Harvest the cells, and store the pellet at -20 &#176;C.</li>
	<li>Resuspend the pellet in 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF. Homogenize and disrupt the cells as described in 2.4 and 2.5.</li>
	<li>Immediately centrifuge the cells lysate at 10,000 x g for 20 min. Discard the pellet. Filter the supernatant with a B&#252;chner funnel equipped with 3 layers of paper filter. Apply gentle vacuum. To avoid excessive foaming, keep the vacuum Erlenmeyer flask on ice during filtration.</li>
	<li>Determine protein concentration according to Bradford14.</li>
</ol>
4. Gel Filtration Chromatography
<ol>
	<li>Equilibrate a water-jacketed 16x70 (200) gel filtration column with 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8. Load onto the column the soluble protein extract. For optimal resolution, use a 1 ml sample loop. Perform the chromatography at 0.6 ml/min. Keep column temperature at 4 &#176;C throughout.</li>
	<li>Collect 0.9 ml fractions and analyze them by SDS-PAGE. Transfer 20 &#956;l of each relevant fraction to Eppendorf tubes containing 60 &#956;l of H2O and 20 &#956;l of loading buffer (250 mM Tris-HCl pH 6.8, 10 mM &#946;-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol, 0.25% (w/v) bromophenol blue) and boil for 5 min. Load 18 &#956;l of each sample and perform the electrophoresis at 140 V for about 1.5 hr.</li>
	<li>Determine the 3&#8217;-5&#8217; exonuclease and the DNA polymerase activity of each fraction in 96-well microplates. Assay the exonuclease activity using thymidine 5&#8217;-monophosphate p-nitrophenyl ester (pNP-TMP) as substrate15. Estimate DNA polymerase activity using the PPX (Pyrophosphatase, Purine nucleoside phosphorylase, Xanthine oxidase) enzyme-coupled assay16 and 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) as the electron acceptor16.</li>
</ol>
5. Mutation Analysis of Populations Co-expressing the Wild-type &#949; Subunit of E. coli DNA Polymerase III and the Mutagenic &#949;D12A Variant
<ol>
	<li>Transfer a single colony of E. coli TOP10 containing the pBAD-&#949; and the pGOOD1-&#949;D12A11 vectors to 1 ml of LB-antibiotics medium. Incubate O/N at 37 &#176;C.</li>
	<li>Dilute the pre-culture 1:250 in 3 flasks containing fresh medium (10 ml), add the appropriate inducers (arabinose, IPTG, or arabinose and IPTG, 1 mM each) and incubate at 37 &#176;C for 8 hr. Prepare in parallel non-induced cultures.</li>
	<li>Collect aliquots of 1 ml, then dilute 1:500 in new flasks containing fresh medium (10 ml) supplemented or not with the appropriate inducers. Incubate O/N at 37 &#176;C.</li>
	<li>Repeat steps 5.2 and 5.3 and collect aliquots of 1 ml.</li>
	<li>Determine the number of generations occurred in each culture. Transfer on LB plates, 100 &#956;l of appropriate serial dilutions of inoculum and culture. Incubate O/N at 37 &#176;C. Count the colonies on the LB plates, and calculate the log of the number of cells present in the inoculum (logI) and in the culture at the end of growth (logC). To determine the number of generations, use the formula: (logC&#8211; logI)/0.301.</li>
	<li>Centrifuge at 5,000 x g for 20 min and resuspend the cells in 1 ml of 50 mM Tris-HCl pH 7.6, 50 mM NaCl. To permeabilize the cells, add 2-3 drops of chloroform and vortex for 20 sec.</li>
	<li>Determine the &#946;-glucosidase activity of each aliquot in a 96-well microplate, using p-nitrophenyl-&#946;-D-glucopyranoside (PNPGluc) as substrate. Add to each well 100 &#181;l of permeabilized cells and 100&#160;&#181;l of substrate (16 mg/ml stock solution in H2O). Take care to avoid air bubbles in the wells. Read the Absorbance at 420 nm, using a microplate reader and the appropriate filter.</li>
</ol>

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The authors declare that they have no competing financial interests.

Proteins can be intrinsically disordered, featuring regions whose tertiary structure is not restricted to a limited number of conformations25. These disordered proteins are usually prone to aggregation25, and their isolation and characterization might represent a difficult task. The &#949; subunit of E. coli DNA polymerase III features two distinct domains26,27, namely: i) the N-ter domain, bearing the 3&#8217;-5&#8217; exonuclease activity, and competent in binding the &#952; su...

The introduction of overexpression technologies boosted either the biochemical studies of low-copy number enzymes and the industrial production of pharmacologically active proteins (e.g.,&#160;insulin). Since the advent of these technologies, significant advances have been achieved to increase the yield and quality of recombinant proteins. In addition, prokaryotic1,2 and eukaryotic3 overexpression systems were developed over the years, offering useful alternatives to the &#8220;work horse&#8221; of protein biotechnology, i.e., Escherichia coli. In particular, the availability of alternative platforms to E. coli led to the production of recombinant peptides or proteins bearing post-translational modifications. However, it should be mentioned that E. coli still represents the organism of choice for recombinant protein production. This is due to several factors, among which the most relevant can be considered: i) the availability of quite a number of overexpression systems (expression vectors and strains) for E. coli1,2; ii) the short generation times, and high biomass yields, of E. coli in a variety of rich and synthetic media; iii) the facile manipulation either at the biochemical and at the genetic level of this microorganism; iv) the isolation of strains capable of the production of toxic proteins4; v) the construction of strains featuring homogeneous induction at the population level5,6. In addition, it was recently shown that expression systems suitable for the production in E. coli of post-translationally modified proteins can be devised and constructed2.
At present, protein overexpression is mainly used to obtain monomeric or homo-oligomeric proteins, whose hypersynthesis can be performed with a single gene cloned into an appropriate plasmid. However, attention was recently paid to the construction of E. coli protein co-expression systems, challenging the production, in vivo, of hetero-oligomeric complexes2. Interestingly, early experiments of protein co-expression addressed the inter-species assembly of large and small subunits of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase7,8, and the association of truncated and full-length forms of HIV-1 reverse transcriptase9. These pioneering studies demonstrated that protein co-expression represents a powerful alternative to traditional in vitro reconstitution. In addition, protein co-expression in E. coli was used to produce different proteins bearing post-translational modifications2, to obtain proteins containing unnatural amino acids2, and to increase the yield of overexpressed membrane proteins2. Moreover, the potential of protein co-expression as a tool to confer to E. coli competence in protein secretion is under active investigation2.
Two main strategies of protein co-expression in E. coli can be pursued: i) the use of a single plasmid to host the different genes to be overexpressed; ii) the use of multiple plasmids in single cells to co-express the target proteins. In the first case, the criteria for the choice of plasmid do not differ from those of traditional single protein overexpression experiments, although particular plasmids containing tandem promoter/operator elements were constructed for co-expression10. This first approach is therefore quite simple. However, it should be mentioned that the use of a single plasmid to co-express different proteins faces two major difficulties: i) the molecular mass of the vector increases with the number of hosted genes, limiting the number of co-expressed proteins; ii) when multiple genes are cloned under the control of a single promoter, polarity can decrease the expression of the genes distal from the promoter. The use of dual or multiple plasmids in single E. coli cells has to accomplish the compatibility of the vectors of choice, therefore imposing constraints to the eligible combinations of plasmids. However, this second co-expression strategy features the advantage of containing the molecular mass of vectors, and limits polarity. We recently constructed a protein co-expression system designed to facilitate the shuttling of genes between the co-expression plasmids11. In particular, we constructed the pGOOD vectors series, the relevant features of which are: i) a p15A origin of replication, to provide compatibility of the pGOOD plasmids with the commercial vectors containing the ColE1 origin (e.g., the pBAD series12); ii) a tetracycline-resistance cassette; iii) the presence of lac-derived regulatory elements, i.e., the Promoter-Operator(O1) couple and the lacIq gene. Using an appropriate pBAD-pGOOD couple, we were able to overexpress the catalytic core of E. coli DNA polymerase III, composed of three different subunits, i.e., &#945; (the 5&#8217;-3&#8217; polymerase), &#949; (the 3&#8217;-5&#8217; exonuclease) and &#952; (stabilizing &#949;)13. In particular, we demonstrated that the co-expression of the &#945;&#949;&#952; complex was strictly dependent on the addition to the E. coli culture medium of both IPTG and arabinose, triggering overexpression from pGOOD and pBAD, respectively (Figure 1A).
In the present report, we illustrate how protein co-expression can efficiently solve difficulties linked to the poor solubility of a protein complex subunit. In addition, we show how in vivo protein complementation tests can be performed, and we finally report on the use of protein co-expression to tune mutation frequency in E. coli. To this aim, we used pGOOD-pBAD couples suitable to illustrate relevant examples of each case study.

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the multifaceted benefits of protein co-expression in escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The &#949; subunit of E. coli DNA polymerase III consists of 243 amino acids and features poor solubility17,18, unless the residues 187-243 are deleted17. However, we have previously shown11 that the co-expression of full-length &#945;, &#949;, and &#952; subunits yields soluble DNA polymerase III catalytic core (Figure 1). In particular, using the pBAD-pGOOD co-expression system, we demonstrated that: i) the overexpression of &#945; and &#949; subunits can be in...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

Research

JoVE Journal

Biology

11.8K 次观看.  University of Bologna. Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells. 

视频: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.