3.4: Prepare expression system - Biology

3.4: Prepare expression system - Biology

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Now that we have prepared DNA encoding your mutant inverse pericams, we would like to actually produce the proteins. Today you will extract DNA from the XL1-Blue cells, prepare it for analysis, and transform your IPC mutant plasmids into a new bacterial system that can produce the protein directly.

The bacterial expression vector we are using (Invitrogen pRSET A, B, & C) contains the bacteriophage T7 promoter. This promoter is active only in the presence of T7 RNA polymerase (T7RNAP), an enzyme that therefore must be expressed by the bacterial strain used to make the protein of interest. We will use the BL21(DE3)pLysS strain, which has the following genotype: F-, ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS (CamR). In BL21(DE3), T7RNAP is associated with a lac construct, and its expression is under the control of the lacUV5 promoter. Due to the action of the lac repressor (lacI gene), the polymerase will not be produced except in the presence of lactose or a small-molecule lactose analogue such as IPTG (isopropyl β-D-thiogalactoside). To further reduce 'leaky' expression of the protein of interest (in our case, inverse pericam), the pLysS version of BL21(DE3) contains T7 lysozyme, which also inhibits basal transcription of T7RNAP. This gene is retained by Chloramphenicol selection, while the pRSET plasmid itself (and thus inverse pericam) is retained by Ampicillin selection - as you learned last time.

To isolate the inverse-pericam-containing pRSET plasmid from the overnight cultures, you will perform what is commonly called a "mini-prep." This term distinguishes the procedure from a "maxi-" or "large scale-prep" which involves a larger volume of cells and additional steps of purification. The overall goal of each "prep" is the same-to separate the plasmid DNA from the chromosomal DNA and cellular debris, allowing the plasmid DNA to be studied further. In the traditional mini-prep protocol, the media is removed from the cells by centrifugation. The cells are resuspended in "Solution I" which contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. A solution of sodium hydroxide and SDS is then added. The base denatures the cell's DNA, both chromosomal and plasmid, while the detergent dissolves the cellular proteins and lipids. The pH of the solution is returned to neutral by the acetic acid and potassium acetate in "Solution III." At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. In addition, the DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The plasmid DNA renatures normally and stays in solution, effectively separating plasmid DNA from the chromosomal DNA and the proteins and lipids of the cell.

Once you have the plasmid DNA isolated, you can prepare it for sequencing and gel analysis, as well as use it immediately for transformation. In order to transform BL21(DE3) cells with your mutant IPC plasmids, you will first have to make the cells competent, i.e., able to efficiently take up foreign DNA. With the XL1-Blue strain, we used commercially available competent cells that did not need further treatment prior to DNA addition. Today, you will make chemically competent cells yourself using calcium chloride, then incubate them with plasmid DNA and heat shock them as before prior to plating. Tomorrow, the teaching staff will pick colonies and set up liquid overnight cultures from your transformed cells. Next time, you will add IPTG to these liquid cultures to induce expression of your mutant proteins, which you will then isolate and characterize. Much of this process is summarized in the figure above.


Part 1: Prepare Competent BL21(DE3) Cells

  1. Pick up one 5 mL tube of BL21(DE3) cells. These cells should be in or close to the mid-log phase of growth, which is indicated by an OD600 value of 04.-06.
  2. Measure the OD600 value of a 1:10 dilution of your cells (use a total volume of 600 μL). If the cells are not yet dense enough, return them to the rotary shaker in the incubator. Remember to balance with another tube! As a rule, your cells should double every 20-30 min.

  1. Once your cells have reached the appropriate growth phase, pour them into eppendorf tubes. Spin down 3 tubes of ~ 1.5 mL each for 1 min at max speed (~16,000 rcf/13,000 rpm), aspirate the supernatants, and resuspend in an equal volume of ice-cold calcium chloride (100 mM). Note: you can balance these tubes in the centrifuge with three-way symmetry.
    • If you are nervous about pouring the liquid, you can use your P1000 to pipet 750 μL into each eppendorf twice. Either way, the eppendorf should be quite full when you try to close the cap. You can wear gloves to keep the bacteria from splashing your skin or you can wash your hands after closing all the caps.
    • You may find it easiest to resuspend the cells in a small volume first (say, 200 μL), then add the remaining volume of CaCl2 (e.g., in two steps of 650 μL) and invert the tubes to mix.
  2. Spin again for 1 min. The resultant pellets should occur as streaks down the side of the eppendorf tube, so be very careful not to disturb the cells when aspirating.
  3. This time, resuspend each pellet in 100 μL of CaCl2, then pool the cells together in one tube.
  4. Incubate on ice for 1 hour. (You might work on parts 2, 4, and 5 of today's protocols now, as well as assemble the materials for part 3.)
  5. Meanwhile, label four eppendorfs and pre-chill them on ice. The labels should indicate a (-) no DNA control, a (+) M124S transformation control, and your two mutant candidate transformations (X#Z -1 and -2).

Part 2: DNA Extraction (mini-prep)

  1. Pick up your two candidates cultures, growing in the test tubes labeled with your team colour. Label two eppendorf tubes to reflect your mutations and candidates (X#Z-1, X#Z-2).
  2. Vortex the bacteria and pour ~1.5 mL of each candidate into the appropriate eppendorf tube.
  3. Balance the tubes in the microfuge, and then spin them for one minute.
  4. Aspirate the supernatant, as shown above, removing as few cells as possible.
  5. Resuspend the cells in 100 μL of Solution I, changing tips between samples.
  6. Prepare Solution II by mixing 250 μL of 2% SDS with 250 μL of 0.4M NaOH in an eppendorf tube. Add 200 μL of Solution II to each sample and invert the tubes five or six times to mix. In some cases the samples may appear to "clear" but don't worry if you don't see a big change.
  7. Place the tubes on ice for five minutes.
  8. Add 150 μL of Solution III to each tube and immediately vortex the tubes for 10 seconds with your vortex set at the highest setting. White clumps should appear in the solution after you vortex it.
  9. Place the tubes in the room temperature microfuge and spin them for 4 minutes.
    • While the tubes are spinning, label another set of eppendorf tubes with the candidate names and your team color.
  10. A white pellet should be visible when you remove your tubes from the microfuge. Use your P1000 to transfer 400 μL of each supernatant to the appropriate clean eppendorf tube. It's OK to leave some of the supernatant behind. Avoid transferring any of the white pellet.
  11. Add 1000 μL of room temperature 100% ethanol to each new tube. The tubes will be quite full. Close the caps and invert the tubes at least five times to thoroughly mix the contents.
  12. Microfuge the samples for 2 minutes. It is important to orient your tubes in the microfuge this time since the pellets from this spin may be barely visible.
  13. Remove the supernatants using your P1000, taking care not to disturb the pellet of plasmid DNA that is at the bottom of the tube, and put them in a 15 mL conical waste collection tube. Remove as much of the supernatant as possible, but you do not need to remove every drop since you will be washing the pellet in the next step.
  14. Add 500 μL of 70% ethanol to each pellet. You do not need to fully resuspend the pellet, but you might invert or flick the tube a few times. Spin the samples one minute, orienting the tubes in the microfuge so you will know where to find the pellet.
  15. Immediately remove the supernatant with your P1000, making sure to keep the tip on the side of the tube that doesn't have your pellet. Remove as much liquid as possible, using your P200 set to 100 μL, to remove the last few droplets and/or to streak them up the side of the tube to promote evaporation.
  16. To completely dry the pellets, place your rack in the hood with the caps open for ~ 10 minutes. When the pellets are completely dry, add 50 μL of sterile water to each sample and vortex each tube for 2 X 30 seconds to completely dissolve the pellets. The liquid can be brought back to the bottom of the tubes by spinning them in the microfuge for a few seconds. Store the DNA on ice.

Part 3: Transform BL21(DE3) with Mutant DNA

  1. Prewarm and dry 4 LB+Amp/Cam plates by placing them in the 37°C incubator, media side up with the lids ajar. You will perform one transformation for each of your four samples.
  2. When your competent cells are ready, aliquot 70 μL of cells per pre-chilled eppendorf.
  3. Add 2 μL of the appropriate DNA to each tube. Remember, you are testing plasmid DNA that was prepared from two different colonies for your X#Z mutant, along with DNA from a colony that is already known to be M124S. You will also perform a no DNA control.
  4. Flick to mix the contents and leave the tubes on ice for at least 5 minutes.
  5. Heat shock the cells on the 42°C heat block for 90 seconds exactly and then put on ice for two minutes.
  6. Move the samples to a rack on your bench, add 0.5 ml of LB media to each one, and invert each tube to mix.
  7. Incubate the tubes in the 37°C incubator for at least 30 minutes. This gives the antibiotic-resistance genes some time be expressed in the transformed bacterial cells.
  8. While you are waiting, prepare 3 large glass test tubes containing LB+Amp/Cam, and label them with your team color and sample name. (You can also finish part 5 of the protocol if you have not yet done so.)
    • Both Amp and Cam should be used at 1:1000, and the total volume in each tube should be 2.5 mL.
  9. Also prepare 4 eppendorf tubes containing 180 μL of LB each. You will use these to dilute your transformed cells 1:10 when you retrieve them from the incubator.
    • If you label these tubes with stickers rather than directly on the cap, you can then transfer each sticker to the appropriate plate as you go, saving one labeling step.
    • Note that we are reducing the cell concentration because miniprep DNA is much more concentrated than the DNA resulting from mutagenesis; it also does not require repair, further increasing the transformation efficiency.
  10. Plate 200 μl of each (1:10 diluted) transformation mix on an LB+Amp/Cam plate.
    • Safety reminder: After dipping the glass spreader in the ethanol jar, then pass it through the flame of the alcohol burner just long enough to ignite the ethanol. After letting the ethanol burn off, the spreader may still be very hot, and it is advisable to tap it gently on a portion of the agar plate without cells in order to equilibrate it with the agar (if it sizzles, it's way too hot).
  11. Once the plates are done, wrap them with colored tape and incubate them in the 37°C incubator overnight. One of the teaching faculty will remove them from the incubator and set up liquid cultures for you to use next time.

Part 4: Count Mutant Colonies

When you have a spare moment today, count the colonies that arose on your transformed XL1-Blue plate, as well as on your positive and negative control plates and those of the teaching faculty. Does the negative control have any colonies? How does your mutation efficiency compare to that of the positive control? To that of the teaching faculty's positive control? Please put your colony counts on today's Talk page.

Part 5: Prepare DNA for Evaluation

Diagnostic Digests

You will perform diagnostic digests on the following samples: the inverse pericam parent plasmid (pRSET-IPC), a known mutant pRSET-M124S, and two candidates for your X#Z mutation. "Digest 1" (D1) will be used to show that pRSET-M124S contains the correct mutation, and "Digest 2" (D2) will be used to test if your candidates do. Thus, you will need enough D1 mixture for two reactions (IPC and M124S), and enough D2 mixture for three reactions (IPC and the two X#Z candidates). To avoid pipetting very small volumes of enzymes, and in order to have at least a little extra of each reaction (so you don't run out due to pipetting error), make enough of each digest for four reactions.

The table below is for one reaction and assumes that each digest will consist of a single enzyme. If you decide to use two enzymes for your mutant digest, you should also set up single-enzyme digests to be run as controls. Please see the teaching faculty for assistance.

If you are using the enzyme BseRI, you should triple the amount of enzyme in that digest due to its low stock concentration.

Plasmid DNA4 μL4 μL
10X NEB buffer2.5 μL of buffer #_____2.5 μL of buffer #_____
Enzyme0.25 μL od _AccI_0.25 μL of _____
H2OFor a total volume of 25 μL
  1. Prepare a reaction cocktail for each of the above reactions (digest 1 and digest 2) that includes water, buffer and enzyme. Prepare enough of each cocktail for 4 digests. Leave the cocktails on ice.
  2. Aliquot 4 μL of the appropriate plasmids into five well-labeled eppendorf tubes. The labels should include the plasmid name, the enzyme(s) to be added and your team color.
  3. Add 21 μL of the appropriate cocktail to each tube. Flick the tubes to mix the contents, touch-spin, then incubate the mixtures at 37°C for at least one hour.
    • While your samples are digesting, you can return to Part 3 of the protocol.
  4. Before leaving lab today, please add 2 μL of loading dye to each of the digests you have assembled. You should also prepare undigested samples of parent IPC and each mutant candidate, containing 4 μL of plasmid, 21 μL of water, and loading dye. We will store the digests and the remaining DNA at –20°C.

Sequencing Reactions

As we will discuss in lab today, sequencing reactions require a primer for initiation. Legible readout of the gene typically begins about 40-50 bp downstream of the primer site, and continues for ~1000 bp at most. Thus, multiple primers must be used to fully view genes > 1 Kbp in size. How many basepairs long is inverse pericam? (Try doing a Word Count on this sequence document (DOC) Luckily, we only care about the back end of IPC, i.e., the part containing calmodulin. To be more precise, if the mutation you incorporated occurs later than the 20th residue of calmodulin, set up your reactions with only the "reverse" primer. If your mutation is upstream of CaM-20, you should set up one reaction with the reverse primer and one with the forward, per each candidate. As for M124S, everyone will be given the same sequencing data to analyze, because you are all working with DNA from the same candidate.

The recommended composition of sequencing reactions is 200-500 ng of plasmid DNA and 3.2 pmoles of sequencing primer in a final volume of 12 μL. The miniprep'd plasmid should have ~1 μg of nucleic acid/μL but that will be a mixture of RNA and DNA, so we will guess at the amount of plasmid DNA appropriate for our reactions. If you are setting up reactions with both the forward-reading and the reverse primers, do not mix the two primers together in one tube!

For each reaction, combine the following reagents in an eppendorf tube:

  • 2 μL of your plasmid DNA candidate
  • 18 μL of the primer solution on the teaching bench, which (per 18 μL) contains
    • 5.3 μL of sequencing primer at 1 pmol/μL
    • 12.7 μL sterile water

Mix each solution by pipetting and then transfer 12 μL to an 8-PCR-tube strip. Keep track of which sample is in which tube (A-H), and label your tubes on both the side and the top according to the table below. The teaching faculty will turn in the strips at the MIT Biopolymers Laboratory for sequencing.


For Next Time

  1. The vector pRSET has several properties that make it useful for protein expression and production in bacteria. Some of these were described in today's Introduction. Name 2 other features contained in the pRSET vector and what purposes they serve. (Use your own words to describe the purposes, don't just quote the catalogue.)
  2. BL21(DE3) E. coli are often used for protein expression. In contrast, XL1-Blue E. coli are 'workhorse' cells useful for plasmid propagation. What are the two modified genes in XL1-Blue that make them ideal for this task? It may help you to refer to the cell manual (PDF).
  3. The major assessment for this module will be a research article describing your protein design work. For this assignment, you will write a draft of the introduction to your report. The introduction provides a framework for the story you are about to tell (The Amazing Adventures of a Mutant Calcium Sensor), and thus serves two main purposes. For one, you must provide sufficient background information for a reader to understand the forthcoming results. Just as importantly, you must motivate the audience to keep reading! How? Reveal the significance of the work through connections to both prior scientific accomplishments and future applications. You are welcome to use your own creativity and judgement as to what a good introduction should look like; however, you may find the suggested structure and content below useful.
    • Paragraph 1: most general, "big picture" paragraph. Here you should introduce the reader to the broader context of your experiment and motivate why your research is important. You might address questions such as those below, but you won't necessarily touch on all of them equally or even at all. Be sure to tell a coherent story, not a dense but unconnected list of facts.
      • Why is calcium biologically relevant?
      • What types of natural and synthetic calcium sensors exist and why are they useful?
      • What is protein engineering and by what strategies can it be accomplished?
    • Paragraph 2: "zooming in" somewhat. Now that the reader has a frame for thinking about your research, you can present background information in more depth, including
      • The structure of inverse pericam, and particularly of calmodulin
      • Specific areas of the protein that could be altered (not just the one you chose, but broad categories of modification)
      • Why changing calcium (or M13) affinity or cooperativity could be useful
    • Paragraph 3: most specific, a description of your particular investigation. Finally you can cover topics such as
      • How you chose your specific mutation (and rationale for the M124S mutation) given the local protein structure
      • Your expectations for how these mutations will affect protein function
      • A brief summary of how you intend to assess whether your experiment worked
      • (Later you will add a brief overview of your results and conclusions)
  4. Please complete the midsemester evaluation form (PDF). Complete the questionnaire and then print it out without including your name to turn in. If there is something you'd like to see done differently for the rest of the course, this is your chance to lobby for that change. Similarly, if there is something you think the class has to keep doing, let us know that too.

Reagent List

Microbial Work

  • 100 mM CaCl2, sterile
  • LB (Luria-Bertani broth)
    • 1% Tryptone
    • 0.5% Yeast Extract
    • 1% NaCl
    • autoclaved for sterility
  • Ampicillin: 100 mg/mL, aqueous, sterile-filtered
  • Chloramphenicol: 34 mg/mL in ethanol
  • LB+AMP+CAM plates
    • LB with 2% agar and 100 μg/ml Ampicillin and 34 μg/ml Chloramphenicol

DNA Mininprep

  • Solution I
    • 25 mM Tris pH8
    • 10 mM EDTA pH8
    • 5 mM Glucose
  • Solution II
    • 1% SDS
    • 0.2M NaOH
  • Solution III
    • 3M KAc, pH 4.8

Plasmid Digests

  • Parental plasmid (pRSET-IPC)
  • Mutant plasmids (pRSET-M124S, and your two minipreps)
  • NEB buffers 1-4
  • NEB enzymes

DNA Sequencing Materials

  • Reverse sequencing primer "pRSET-seq" (original stock 100 pmol/μL)
  • Forward sequencing primer "IPC-seq-f1" (original stock 100 pmol/μL)

GAL4/UAS System

T. Xu , . D. Denton , in Methods in Enzymology , 2017

6.1 Expression of Fluorescently Tagged Autophagy Markers In Vivo

The GAL4/UAS system is commonly used in Drosophila to drive expression of a gene of interest ( Brand & Perrimon, 1993 ). Tissue-specific or ubiquitous enhancers drive expression of GAL4 that results in transcriptional activation from the GAL4 binding sites (UAS) to drive expression of a gene of interest, and is widely used for expression of fluorescently tagged proteins and for gene knockdown by RNAi. To assay for autophagy using the fluorescently tagged markers as listed in the above section, a tissue-specific driver GAL4 line can be crossed to the fluorescently tagged UAS lines to generate stocks for further analysis. These lines can then be used to examine mutant alleles or RNAi-mediated gene knockdown.

A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure

Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells exocrine cell types vascular cells Schwann cells quiescent and activated stellate cells and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.

Copyright © 2016 Elsevier Inc. All rights reserved.


Figure 1. A Transcriptomic Map of the…

Figure 1. A Transcriptomic Map of the Human and Mouse Pancreas

Figure 2. The Endocrine Transcriptome Is Readily…

Figure 2. The Endocrine Transcriptome Is Readily Distinguished from the Other Cell Types and across…

Figure 3. Endocrine Transcriptomes Reveal Novel Expression…

Figure 3. Endocrine Transcriptomes Reveal Novel Expression Patterns of Key Genes

Figure 4. Multiple Modes of Pancreatic Stellate…

Figure 4. Multiple Modes of Pancreatic Stellate Cell Activation and Existence of Pancreatic Adult Neural…

Figure 5. Subpopulations of Ductal Cells in…

Figure 5. Subpopulations of Ductal Cells in the Human Pancreas

(A) Differential expression of MUC1,…

Figure 6. Heterogeneity of Beta Cells Reveals…

Figure 6. Heterogeneity of Beta Cells Reveals the Unfolded Protein Response

Figure 7. BSeq-SC Uses Single-Cell RNA-Seq to…

Figure 7. BSeq-SC Uses Single-Cell RNA-Seq to Deconvolve Bulk Heterogeneous Tissue Data and Decouples Disease-Associated…


Direct expression of CL7-tagged Cas9 RNPs from E. coli

To obtain fully formed Cas9 RNPs, the current strategy is to roughly mix Cas9 enzymes and the associated sgRNAs in vitro 17 . The major shortcoming of this methodology is that the sgRNAs are often susceptible to enzymatic degradation during the process by ubiquitous RNases in the environment. Therefore, it is challenging to load high-quality sgRNAs in a sufficient amount to Cas9. We recently established a co-expression method to directly prepare Cas9 RNPs in E. coli 15 . By the approach, the newly synthesized Cas9 enzymes and the transcribed sgRNAs were spontaneously self-assembled within E. coli cells, forming matured Cas9/sgRNA complexes. We found that such kind of self-assembling Cas9 RNPs are very stable which maintain full activity at −20 °C for up to 9 months in the absence of RNase inhibitors. The methodology still has limitations yet, two of which are the relatively low yield and the long purification time.

To increase the yields of Cas9 RNPs, here we introduced a CL7 tag in the N-terminus of original Cas9 16 . The CL7 tag can be easily removed by human rhinovirus (HRV) 3C proteinase recognized cleavage at 16 °C for 3 h 18 . In addition, to prevent contamination of the 3C proteinase in the final sample, an engineered CL7-tagged HRV 3C proteinase was used. The scheme of expression plasmid termed pCold CL7–Cas9 was shown in Fig. 1. The CL7 is a catalytically inactive variant of Colicin E7 (CE7) DNase with a low KD ( ∼ 10 −14 –10 −17 M) toward its binding partner Im7 19 . This CL7/Im7 system has recently been reported to facilitate purification of diverse proteins as well as to enhance their production 16 . According to the design, the sgRNA molecules were abundantly transcribed in E. coli when adding IPTG, while the CL7–Cas9 fusion proteins were simultaneously expressed within E. coli too. The yield of Cas9 RNPs was increased to

40 mg/L when using LB culture medium, which is fourfold higher than incumbent methods. Moreover, we applied the method to produce Cas12a RNPs, and also resulted in a much higher yield (

30 mg/L) than the current method which uses maltose binding protein as the fusion tag 3 . All the gene sequences and plasmid maps are shown in Supplementary Figs. 1–5. The NCBI gene identification for the proteins used in this work are: S. pyogenes Cas9, Gene ID: 901176 F. novicida Cas12a, Gene ID: 2827873 E. coli Colicin E7 DNase (CE7), Gene ID: 20467019. Interestingly, we found that the CL7–Cas9 RNP has a similar endonuclease activity (Supplementary Fig. 6) to Cas9 RNP, indicating that the CL7-tagged variant can be alternatively used for genome editing.

An engineered cold-shock expression vector was harnessed to achieve co-expression of CL7–Cas9 and sgRNA in E. coli. The CL7–Cas9 and sgRNAs were spontaneously self-assembled within E. coli cells to form CL7–Cas9 RNPs. The pure Cas9 RNPs with high stability were prepared by one-step purification and in-column cleavage of CL7 tags using a CL7-tagged HRV 3C protease

One-step purification of Cas RNPs by CL7/Im7 ultrahigh-affinity system

To purify Cas9 RNPs, we previously harnessed a Ni-NTA affinity purification followed by a gel filtration step using the HiLoad 26/60 Superdex 200 column (GE, USA) 15 . During the multistep purification, a large number of Cas9 enzymes might be lost. In addition, two or more days are needed to prepare Cas RNPs. Herein, the introduction of an ultrahigh-affinity CL7/Im7 system 16 helped us achieving one-step purification of Cas RNPs within half a day (Supplementary Table 1, see Supplementary Information for comparative details). Compared with the Cas9 RNPs purified by Ni-NTA affinity column, the purity of Cas9 RNPs obtained by Im7 column was increased from

89% based on the gray scanning analysis (Fig. 2). The various bands visible on the gel for Cas RNPs purified by Ni-NTA were proteins from E. coli itself. The purity of target Cas RNPs can be improved by gradient elution using different concentrations of imidazole follow by a second purification step using gel filtration. Importantly, reproducible results were observed for preparation of Cas9 RNPs with different sgRNAs (Supplementary Fig. 7). In addition, there were no batch-to-batch variations regarding production of the same Cas9 RNPs. Notably, the Im7 affinity column was simply prepared by ourselves through coupling the recombinant Im7 enzymes to agarose beads, and can be repeatedly regenerated without losing its binding affinity to the CL7 affinity tag 16 . So, the high expense of Ni-NTA agarose (

2000 USD for 100 mL from Qiagen, USA) can be saved, making our method very cost-effective. Alternatively, the Im7 ligated agarose beads can be purchased from TriAltus bioscience (Birmingham, AL, USA).

a A total of 12% SDS–PAGE of Cas9 RNP (10 μg, red square) and Cas12a RNP (5 μg, blue square) purified by Ni-NTA affinity column or by Im7 column. The original uncropped gels were shown in Supplementary Fig. 7. b The target Cas RNPs’ purity was validated from three individual batches of purification by gray scanning analysis (ImageJ) of the SDS–PAGE. Data are shown as the mean ± SD

The CRISPR/Cas RNPs prepared by incumbent methods 17 are often unstable which might loss activities within weeks due to the digestion of sgRNAs, even in the presence of RNase inhibitors. By our method, none of RNase inhibitors are yet required in the whole purification and storage processes. The Cas9 RNPs were found very stable which can be stored at −20 °C for 9 months without activity changes. To explain the observations, we proposed that the sgRNAs transcribed in vivo within E. coli could somehow tightly bind to the nascent Cas9, helping Cas9 fold into a stable conformation which protects sgRNAs from nuclease-mediated degradation.

Cleavage of vectors by Cas9 RNPs as artificial restriction endonucleases

Restriction enzymes are essential genetic tools for recombinant DNA technology that have revolutionized modern biological research since the early 1970s 20 . So far, there are over 250 commercially available restriction endonucleases for routine uses in thousands of laboratories around the world 21 . Most of them only recognize short-DNA sequences (typically

6 or 8 bp), which limits their applications in particular use such as seamless DNA cloning. Recently, Wang et al. have successfully employed Cas9 enzymes as artificial restriction enzymes (AREs) that combined with Gibson assembly to accomplish seamless DNA cloning 22 . Yet, it is still difficult to prepare AREs in a sufficient amount at low cost and short time, challenging their wide applications instead of restriction enzymes. To our knowledge, our aforementioned method addressed this issue for the first time, enabling timely and inexpensive production of Cas RNPs.

As known, Cas9 recognizes a

20 bp target sequence with a required downstream NRG (where R = G or A) PAM and induces a site-specific double strand break 23,24 . We searched all PAM sites at the multiple clone sites in popularly used vectors, such as pET28a (+) and pcDNA3.1 (+) (Fig. 3). Taking pcDNA3.1(+), for example, it has 12 restriction enzyme sites and 19 PAM sites in the same region. Even better, we can simply introduce new PAM sites at any desired location in the vectors. It is, therefore, more than enough to generate a sufficient amount of Cas9 RNPs instead of restriction enzymes. More importantly, the multiple PAM sites can facilitate researchers to choose specific sites for molecular cloning in certain situations that the restriction enzymes were forbidden. For instance, we cannot use a restriction enzyme if its recognizing sequence exists both in cloning DNA and vector. Nevertheless, we do not have to worry about the issue when using Cas9 RNPs, because there is a very low probability that the same 20 bp recognizing sequence resides both in cloning DNA and vector. At present, we have prepared a series of Cas9 RNPs as AREs instead of the commercial restriction enzymes for molecular cloning in our laboratory.

a Cleavage of plasmids including pCDNA3.1(+) and pET28a(+) at MCS by two Cas9 RNPs. b The results of Cas9 RNPs cleavage detected by 0.8% agarose page. The cleavage sites were indicated with arrows and numbered in Fig. 3a corresponding to the lanes in Fig. 3b

In vitro nuclease cleavage and in vivo genome editing by Cas RNPs

To determine the in vitro nuclease activity of Cas RNPs, we found that 300 ng plasmids were fully cleaved in less than 30 min at 37 °C when adding 200 ng of Cas RNPs (Fig. 4a), including Cas9 RNP, Cas12a RNP, and CL7–Cas9 RNP. The nuclease activity of produced Cas RNPs is comparable to the commercial CRISPR/Cas enzymes, which indicates that our method can be industrially applicable to produce CRISPR/Cas RNPs.

The endonuclease activity assays of purified Cas RNPs. a In vitro cleavage on the target plasmid I (single cleavage site) by Cas9 RNPs or CL7–Cas9 RNPs, as well as on the target plasmid II (two cleavage sites) by Cas12a RNPs. b Delivery of Cas9 RNPs and donor ssDNA in BFP-HEK293 cells can induce HDR-mediated genome editing which can convert them into GFP-HEK 293 cells. c The bright-filed and fluorescent images of BFP-HEK293 cells after delivery of Cas9 RNPs (left), donor ssDNA only (middle), and Cas9 RNPs together with ssDNA donor (right) by lipofectamine CRISPRMAX in 48 h later. Scale bars: 100 μM. d The HDR efficiency was determined by GFP expression due to BFP editing according to the flow cytometry data (Supplementary Figure 8), including the prepared Cas9 RNPs in this work with ssDNA donor (red), ssDNA donor only (blue), and Cas9 enzyme). Data are shown as the mean ± SD

To illustrate the efficiency of in vivo homology dependent repair (HDR)-mediated genome editing by Cas RNPs, we constructed an engineered blue fluorescent protein (BFP)-expressing HEK293 cell line as the reporter system 25 . When correct genome repairing occurred after co-delivery of Cas9 RNPs and a 70 nt ssDNA donor (see the sequence in Supplementary Information) into cells by lipofectamine CRISPRMAX (Fig. 4b), the BFP-HEK293 cells would be converted to green fluorescent protein (GFP)-expressing cells. The HDR efficiencies were estimated from the flow cytometry data (Supplementary Fig. 8) and shown in Fig. 4d. The HDR efficiency of Cas9 RNPs produced by our method is

19%, which is 1.8 times higher than incumbent methods (

11%). In contrast, none of HDR efficiency was observed when delivering Cas9 RNPs individually and

1% of HDR frequencies were obtained when transferring ssDNA donor only. Collectively, these results indicate that the prepared Cas9 RNPs have profound gene-editing efficiency in vitro and in vivo.


Perfect combination of conventional REL strategy and modern SSR technology confers obvious advantages to BioVector. (1) Exchangeable and Efficient. A gene and a promoter can be easily assembled together to fulfill expressing a gene from a temporal-spatio promoter with different intensity, especially overexpressing genes under the control of native promoters (2) Flexible. GECs, PECs, and BDVs can be ad arbitrium modified with ongoing demands (3) Practical and Versatile. BioVector can be applied to almost all fields in functional genome research of various plants (4) Universal and Time-/Labour-Saving. GECs can be efficiently applied to any plant, yeast, and E. coli destination vectors sharing corresponding SSR sites, and it is possible to construct a worldwide library as shared community resource for GECs, PECs and BDVs (5) Seamless fusion. It is possible to make seamless fusion between a protein and a tag, rather than to introduce a detrimental SSR spacer as the widely-used Gateway recombination system does (6) Broad application and interest. The idea of BioVector can also be applied to similar study in animals and yeast.

Student Evaluation

The student feedback on this course has been very positive, and the original goals set out when this course was redesigned seems to have been achieved. In Table 3, selected evaluation questions from the Fall 2016 are shown. The overall score for the module is 4.9 out of 5.0. The students further give high ratings for the learning outcome, the one-project idea, higher engagement, more research relevance, and the depth and breadth of the course. They have also gained more confidence to use these methods themselves. When the students are asked what generic skills they have learned through the course, they point out academic writing, interacting with students and teachers, team work, and time management. These are important skills for the students when they are continuing their work with a master thesis in a research group. Several of the above mentioned outcomes could also coincide with important goals in CUREs-based courses. Regarding the gained scientific skills, stundents acknowledge learning different techniques and being aware of various applications of the methods learned.

Question Score
How would you rate the module? 4.9
How would you rate your learning outcome? 4.6
How would you rate handing-in the report as an article in an article template? 4.5
How do you rate running this part as one large project “From Gene to Structure and Function”, compared to laboratory courses with smaller, separated laboratory exercises instead? 4.5
How engaging was it to run one project compared to more separated laboratory exercises? 4.6
Do you feel this project gave you more insight into research than normal laboratory courses? 4.6
How confident are you in using these methods/topics yourself in the future? 4.0
How well do you feel going in depth in one example protein worked out with respect to opening up and extending for future use of related/similar methods/system, the so-called breadth? 4.7

Protein Expression and Purification Core Facility

To speed up protein production, we have adopted a strategy of parallel expression of a protein from a variety of vectors containing different tags and/or fusion partners, and a variety of E. coli host strains. This approach should not only gain us a lot of time but also result in a larger number of successfully expressed proteins.

The expression strategy consists of the following two sets of experiments:

1. The expression of a protein in a basic E. coli host strain from a variety vectors with different tags and/or fusion partners.

Our first screen is to express a protein in BL21 (DE3) from modified pET-vectors with the following a selection of tags and fusion partners:

Tag fusion partner
N-terminal His6-tag
N-terminal His6-tag thioredoxin
N-terminal His6-tag glutathione-S-transferase (GST)
N-terminal His6-tag maltose binding protein (MBP)
N-terminal His6-tag disulfide oxidoreductase (DsbA)
N-terminal His6-tag NusA
C-terminal His6-tag

2. The expression of a protein from a standard vector in a number of different E. coli host strains.

The choice of the host strains depends more on the nature of the heterologous protein. The following considerations should be made:

  • If the protein contains a high number of rare E. coli codons, it is worthwhile trying to express it in a strain that co-expresses the tRNAs for these rare codons. There are several strains commercially available:
  • If the protein contains one or more disulfide bonds, proper folding is stimulated in host strain with a more oxidizing cytoplasmic environment. Two strains are commercially available from Novagen:

  • If the protein is toxic to the cell, expression in a strain containing the pLysS or pLysE vector tightens regulation of expression systems using the T7 promoter. These vectors express lysozyme, which binds to and inactivates T7 RNA polymerase. Strains are commercially available from different manufacturers.

Our first screen is to express a protein from a modified pET-vector with an N-terminal His6-tag in the following host strains:

Host strain
BL21 (DE3)
BL21 (DE3) pLysS
BL21 (DE3) CodonPlus-RIL (-RP) or Rosetta (DE3)
Origami (DE3)

For the rapid screening of expression levels and protein solubility from the different vectors and in the different strains we have developed a small scale method using chromatography on magnetic beads.

The results of the first screen will be a starting point for further experiments in case no satisfactory expression conditions have been found. How to optimise expression levels, improve protein solubility, improve protein stability, and decrease protein toxicity will be discussed in the other chapters in this website.

Expression method

A typical expression experiment consists of the following step:

  • Picking of a single colony from a freshly streaked plate of the expression host containing the recombinant vector. When the heterologous protein is toxic for the cells, higher expression levels are obtained by using the so-called "plating" method.
  • Growing of a starter culture. Inoculate with the picked colony up to 50 ml of rich medium (such as LB or 2xYT) containing the appropriate antibiotic. When a larger starter culture is required, inoculate 4 ml of rich media with the single colony grow for 4-8 hours at 37°C and use this to inoculate the starter culture.

Do not let cultures grow at 37°C overnight! It is better to grow overnight cultures at 30°C or lower. Alternatively, the culture can be incubated at 37°C until the OD600 is approx. 1. Then store the culture at 4°C overnight. The following morning, collect the cells by centrigfugation, resuspend them in fresh medium and use this to inoculate the main culture.

The use of ampicillin requires special care. The selectable marker, b -lactamase, is secreted into the medium where it hydrolysis all of the ampicillin. This point is already reached when the culture is barely turbid. From here on, cells that lack the plasmid will not be killed and could overgrow the culture (which can be tested using a plasmid stability test). Some possible solutions are:

  • grow overnight cultures at 30°C or lower.
  • spin overnight cultures and resuspend the pellet in fresh medium to remove b -lactamase.
  • use the more stable carbenicillin instead of ampicillin.
  • Inoculation of the main culture and incubation until OD600 reaches 0.4-1. The optimal OD value depends on the culture method and the medium. For flask cultures using LB-medium an OD600of 0.6 is recommended. To increase the growth rate, we carry out the cultures at 37°C until the OD for induction is reached. Then the cultures are cooled to the induction temperature in ice-water.

Remark: For good aeration, don't use more medium than 20% of the total flask volume.

  • Induction of protein expression. Protein expression is induced by the addition of the proper inducer or by changing the growth conditions. From this point on the cells will use most of their resources for the production of the target protein and will not grow much further.

For the most used promoters induction conditions are listed below.

Promoter induction typical condition range
trc (hybrid) addition of IPTG 0.2 mM 0.05 - 2.0 mM
araBAD addition of l-arabinose 0.2% 0.002 - 0.4 %
PL shifting the temperature from 37 to 42°C
T7-lac operator addition of IPTG 0.2 mM 0.05 -2.0 mM

After induction the cultures are incubated from 3 hours to overnight depending on the induction temperature. Guide lines are given below.

Author summary

The key points of this study are two-fold: The first point is the decoding mechanism for cell differentiation. We previously demonstrated the encoding mechanism of cell fate decision information by transient and sustained ERK activation in PC12 cells, and also identified the decoding genes essential for cell differentiation in PC12 cells, including Metrnl, Dclk1, and Serpinb1a, denoted as LP (latent process) genes, which are the decoders of neurite length information. Importantly, the expression levels of the LP genes, but not the phosphorylation level of ERK, correlate with neurite length. Thus, the decoding mechanism of signaling activities by LP gene expression is a key issue for understanding the mechanism of cell differentiation. Here we identified a selective NGF- and PACAP-signaling decoding system by LP gene expression for neurite extension by developing a system identification method. The second point is the modeling. Cells decode information of signaling activation at a scale of tens of minutes by downstream gene expression with a scale of hours to days, leading to cell fate decisions such as cell differentiation. However, no system identification method with such different time scales exists. Here we developed a signal recovery technique in the field of compressed sensing originally developed for image analysis to biological sparse data of different time scales of signaling and gene expression.

Citation: Tsuchiya T, Fujii M, Matsuda N, Kunida K, Uda S, Kubota H, et al. (2017) System identification of signaling dependent gene expression with different time-scale data. PLoS Comput Biol 13(12): e1005913.

Editor: Alexander Hoffmann, University of California Los Angeles, UNITED STATES

Received: July 24, 2017 Accepted: December 1, 2017 Published: December 27, 2017

Copyright: © 2017 Tsuchiya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data files are available from our database (

Funding: This work was supported by the Creation of Fundamental Technologies for Understanding and Control of Biosystem Dynamics, CREST, of the Japan Science and Technology Agency (JST) (#JPMJCR12W3,, and a Grant-in-Aid for Scientific Research on Innovative Areas (#17H06300, #17H06299, TT receives funding from a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Research Fellow (#14J12344, MF receives funding from a Grant-in-Aid for Challenging Exploratory Research (#16K12508, KKu receives funding from a Grant-in-Aid for Young Scientists (B) (#16K19028, SU receives funding from a Grant-in-Aid for Scientific Research on Innovative Areas (#16H01551, HK receives funding from a Grant-in-Aid for Scientific Research on Innovative Areas (#16H06577, KKo receives funding from a Grant-in-Aid for Scientific Research (B) (#15KT0021,, and (C) (#15K00246,, and Grant-in-Aid for Scientific Research on Innovative Areas (#16H01554, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Recombinant Protein Definition

Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins, modify gene sequences and manufacture useful commercial products. The formation of recombinant protein is carried out in specialized vehicles known as vectors. Recombinant technology is the process involved in the formation of recombinant protein.

Recombinant Protein is a protein encoded by a gene — recombinant DNA — that has been cloned in a system that supports expression of the gene and translation of messenger RNA (see expression system). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. Proteins coexpressed in bacteria will not possess post-translational modifications, e.g. phosphorylation or glycosylation eukaryotic expression systems are needed for this.

Recombinant DNA (rDNA) molecules are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure they differ only in the sequence of nucleotides within that identical overall structure. Consequently, when DNA from a foreign source is linked to host sequences that can drive DNA replication and then introduced into a host organism, the foreign DNA is replicated along with the host DNA.
Proteins that result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein will not necessarily be produced. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring of the foreign coding sequence.

Biologics International Corp (BIC) provides our esteemed clients with the rapid, high quality, and cost-effective recombinant protein production services. We offer manufacturing as well as wild type, mutant, and ortholog proteins. Our services could advance your project from gene synthesis to protein expression, purification, and stable cell line construction. Till date, we have successfully delivered 2000+ recombinant proteins (enzymes, cytokines, growth factors, hormones, receptors, transcription factors, antibodies, antibody fragments, etc.), which have been widely used in antibody preparation, enzyme activity assay, in vivo efficacy evaluation, in vitro diagnosis, as well as vaccine screening and other applications.

Choose BIC as your reliable partner for your protein research, and we can help you accelerate your discovery in a timely and cost-effective manner at every step of the way, and at a very affordable price. Contact us today to speak with our protein specialists.

WGCNA: an R package for weighted correlation network analysis

Semel Institute for Neuroscience and Human Behavior, UC Los Angeles (PL),
Dept. of Human Genetics and Dept. of Biostatistics, UC Los Angeles (SH)

Peter (dot) Langfelder (at) gmail (dot) com, SHorvath (at) mednet (dot) ucla (dot) edu

Quick navigation


The WGCNA R software package is a comprehensive collection of R functions for performing various aspects of weighted correlation network analysis. The package includes functions for network construction, module detection, gene selection, calculations of topological properties, data simulation, visualization, and interfacing with external software. Along with the R package we also present R software tutorials. While the methods development was motivated by gene expression data, the underlying data mining approach can be applied to a variety of different settings.

Getting started with R and Weighted Gene Co-expression Network Analysis

Readers wishing to learn about the theory and published applications of WGCNA are invited to visit the WGCNA main page.

R Tutorials

Click here to access the tutorial page.

Further reading

Peter Langfelder occasionally writes about WGCNA features and other topics relating to data analysis. The articles are written for a general audience and try to avoid deep technical details. We also have a few technical reports that discuss a selected deeply technical aspects of the WGCNA methodology - these are more mathematical and targeted primarilly to die-hard statistician geeks.

Automatic installation from CRAN

The WGCNA package is now available from the Comprehensive R Archive Network (CRAN), the standard repository for R add-on packages. Currently, some of the required packages is only available from Bioconductor and need to be installed using Bioconductor's installation tools. The easiest way to do this is

The first command ( install.packages("BiocManager") ) can be skipped if the package BiocManager is already installed.

This will install the WGCNA package and all necessary dependencies. The catch is that this only installs the newest version of WGCNA if your R version is also the newest (minor) version. Users using older versions of R will need to follow the manual download and installation instructions below.

Note for Mac users: CRAN occasionally fails to compile the WGCNA package for Mac OS X. This leads to the error message "Package WGCNA is not available. " when calling BiocManager::install() . If this occurs, please download the binary version from here and follow the installation instructions (or, if you are able to compile packages locally, download the source and install that).

Note of caution: The newest versions of WGCNA is available from CRAN only for the current R version and (usually) one older version. For example, if your R version is 3.2.1 and the current R version on CRAN is 3.5.0, the automatic installation and update will not use the newest version of WGCNA. Please update your R to the newest version or use the manual download below.

Problems installing or using the package? Please see our list of frequently asked questions. Your problem and the solution may already be posted there.

Manual download and installation

Please follow these steps only if the automatic package installation above does not work.

The current version of the WGCNA package will only work with R version 3.0.0 and higher. If you have an older version of R, please upgrade your R.

The WGCNA package requires the following packages to be installed: stats, grDevices, utils, matrixStats (0.8.1 or higher), Hmisc, splines, foreach, doParallel, fastcluster, dynamicTreeCut, survival, parallel, preprocessCore, GO.db, impute, and AnnotationDbi. If your system does not have them installed, the easiest way to install them is to issue the following command at the R prompt:

Please note that GO enrichment calculations in WGCNA are deprecated we recommend using the R package anRichment which provides replacement for WGCNA functions GOenrichmentAnalysis() and userListEnrichment().

If you run an older version of R, the above may not install the newest version of the dynamicTreeCut package. Should you encounter this problem, please manually download and install dynamicTreeCut from this web page.

  • Source for Linux and all users able to compile the package locally: WGCNA_1.69-81.tar.gz
  • Compiled binary Mac OS X: WGCNA_1.69.tgz
  • Comiled binary for Windows running R-3.4.0 or higher:
  • Reference manual in pdf format
  • Quick reference: overview table of most important functions
  • A terse changelog

The package version numbers follow the format packageName_major.minor-revision. Minor versions typically add or change some functionality revisions typically contain bugfixes or minor enhancements.

Should you discover bugs (of which there are most likely plenty), please report them to Peter Langfelder.

Problems installing or using the package

Please see our list of Frequently Asked Questions (and frequently given answers) the solution to your problem may lie there. In particular, you can find answers about spurious Mac errors, compatibility problems when upgrading WGCNA, and others. If you still cannot solve the problem, email Peter Langfelder.

Additional files

Additional file 1

Table S1. Reactions with perturbed keff. A subset of the genome-scale metabolic network was perturbed with respect to keff values, either manually or randomly. (XLSX 17 kb)

Additional file 2

Figure S1. Scree plot for determining number of archetypes. A notable elbow is observed for five archetypes. (PDF 5 kb)

Additional file 3

Figure S2. Parameter estimation procedure. We developed a parallel implementation of a metaheuristic optimization procedure. L-TA: list-based threshold accepting algorithm [37]. Variable definitions: T k , threshold value at iteration k T max , maximum threshold value Z 0 , objective value at current solution Z k , objective value at neighboring solution (generated from current solution) at iteration k. (PDF 189 kb)

Additional file 4

Figure S3. Parameter estimation results. The parallel L-TA optimization procedure successfully estimated model parameters that improved consistency with measured concentration profiles. Seven parallel nodes were used here: 1 local and 6 global nodes (see Additional file 3: Figure S2) for explanation of nodes. (PDF 124 kb)

Additional file 5

Table S2. Lagged cross-correlation values. Cross-correlation values for fixed lag time of 1.2 h. (XLSX 41 kb)