Genetic Engineering

Created: Sep 2021

Last Updated: Jan 2022

1. Introduction

The advances in the field of biology are remarkable, and genetic engineering provides the technology to facilitate those advances. Genetic engineering consists of manipulating and recombining DNA- the molecule storing the information about an organism. The main operations involving DNA are DNA increase, cutting and connecting. Increase (duplication) is performed by the concerned organism itself, cutting is performed by restriction enzymes, and connecting is performed by ligase.

Genetic engineering has been contributing to the progress of the society and improvement of the quality of life in numerous ways. Examples of this include herbicide resistant genetically modified crops, genetic treatment alleviating the symptoms of intractable diseases such as cancer, and iPS cells, which are used in regenerative medicine.

In this experiment, we will use several techniques that are widely used in everyday genetic engineering experiments. Firstly, we will perform transformation of E. coli. Transformation is the process of moving DNA from a certain species to another (genetically different) species in order to induce genetic changes. In the first experiment, E. coli cells are made competent, then a plasmid DNA (pUC19) carrying an antibiotic resistance gene is introduced into those recipient cells (competent cells). After the bacteria have been cultivated overnight, E. coli into which the plasmid has been incorporated form colonies. The newly formed colonies are transferred to liquid medium, cultured, and used in the next experiment.

In the second experiment, we will perform isolation of plasmid DNA from the E. coli cells using the alkaline SDS method. We will separate chromosomal DNA and plasmid DNA and then isolate the CCC state plasmid by alkali processing method. Overall, we will isolate the plasmid DNA from the 2 liquid cultures of E. coli.

The third experiment involves using restriction enzymes, which recognize and cleave a double-stranded DNA at a specific nucleotide sequence. We will use 2 restriction enzymes: HindIII and PvuII.

In the fourth experiment, we will separate the linear double-stranded DNA by agarose gel electrophoresis. By investigating the mobility of the DNA fragments, we will try to determine the size of the specific fragments formed by cleaving the plasmid DNA with restriction enzymes

The purpose of these experiments is to acquire basic knowledge and techniques for conducting genetic research, through learning experimental manipulation methods and analysis methods of genetic engineering experiments that are routinely performed in many laboratories handling genes. Moreover, we will attempt to determine the size of the DNA fragments (in base pairs) obtained though cleavage of pUC19 plasmid DNA using HindIII and PvuII restriction enzymes.

2. Materials and Methods

2.1. Preparation and Transformation of E. coli Receptor Cells

Transferring DNA from a certain species to another (genetically different) species in order to induce genetic changes is called transformation. Adding genetic traits (abilities) not originally possessed by bacteria such as E. coli etc. through incorporating plasmid DNA into them, thus transforming the cells, is an important technology in genetic engineering.

In this experiment, a plasmid DNA carrying an antibiotic resistance gene is introduced into E. coli in order to create an E. coli recombinant that has acquired the antibiotic resistance trait.

In general, plasmids are not incorporated into E. coli in nature. Therefore, it is necessary to change the surface state of E. coli cells by artificial processing so that the plasmid can be introduced. This is called competence, and there are various chemical and physical methods of achieving it. The cells that have been made competent and into which the plasmid can be introduced are called recipient cells (competent cells).

In this experiment, E. coli is made competent by a simple calcium chloride method, and plasmid DNA (pUC19) containing the ampicillin resistance gene is introduced into the obtained cells. As the result, E. coli which were originally sensitive to ampicillin become resistant to it. After the bacteria have been cultivated overnight, E. coli into which the plasmid has been incorporated form colonies. Those colonies are transferred to liquid medium, cultured, and used in the next experiment (plasmid isolation).

Equipment

• Micropipettes

• Micropipette tips

• Tubes

• Clean bench

• Centrifuge

• Gas burner

• Spreader

• Incubator

• High temperature water tank

Reagents

• E. coli DH5α strain

• pUC19 (plasmid)

• 0.1 M CaCl2

• SOC medium

• 0.9% NaCl

• LB medium agar plate

• LB medium agar plate containing 100 mg/L ampicillin

• Liquid LB medium containing 100 mg/L ampicillin

Precautions

Do not discard the bacterial solution in the sink after the experiment. Place the microtubes and tips in the waste bin on the laboratory bench. Dispose of the bacterial cell suspension liquid and bacterial cell culture waste into a dedicated cell waste container provided in the laboratory. It is desirable to perform the competence procedure aseptically in order to prevent contamination, but in this case, the experiment is performed on each experiment desk up to step 3). In order to increase transformation efficiency, it is important to maintain low temperature at all times during the competence process, so the samples should be stored on ice while not used.

When diluting a solution, make sure to suspend (mix) it before and after dilution, as the cells gradually precipitate.

Procedure

1) Bacterial Cultivation

Pickup single colony of E. coli cells from a LB plate, inoculate it into 10 ml LB broth, and incubate for 16-20 hours at 37°C while shaking. Transfer 1 ml of culture into 100 ml of fresh LB broth in a 500 mL flask and incubate for 4 hours at 30°C while shaking. Collect 5 ml of culture to disposable, ice-cold polypropylene tube. Cool the culture on ice for over 20 minutes, Harvest the cells by centrifugation at 7,000 rpm for 5 minutes at 4°C.

2) Washing and Resuspending the Bacteria

Prepare an icebox and put the tube containing centrifuged culture inside. Carefully decant the media from the cell pellets. Suspend each pellet in 2 ml of ice-cold 0.1 M CaCl2 and store on ice until centrifugation. Recover the cells again by centrifugation at 7,000 rpm for 3 minutes at 4°C. Decant the fluid from the cell pellets. Add 0.3 ml of ice-cold 0.1 M CaCl2 and resuspend the cell pellets. Collect 100µL of suspended cells into a 1.5 ml tube and store on ice.

3) Starting the Transformation

Add 5µL of plUC19（10ng/µL）and mix gently by pipetting。Store the tube on ice for 15 minutes.

4) Heatshock, Heat removal, Addition of LB

Incubate the culture at 42°C for 30 seconds (exactly) in a water bath. Do not shake the tube during incubation。Rapidly transfer the tube to an icebox and chill for 3 minutes。Add 900µL of LB medium to the tube. Incubate the culture at 37°C for 15 minutes in water bath in order to enable the bacteria to express the ampicillin resistance gene encoded by pUC19.

5) Dilution of the transformant, Application, Cultivation

To dilute the cells（105 times）, prepare five tubes with 900µL of 0.9％ NaCl。Dilute the transformant cells。Drop 100µL of x105 diluted cells onto a LB medium agar plate and gently spread the cells. Invert the plate and incubate at 37°C.

6) Transplantation (next day)

Working inside of a clean bench, inoculate the colony from the obtained LB plate into a liquid medium. To do this, collect some bacteria on a sterilized toothpick and place the toothpick inside of a test tube containing 2 mL of LB liquid medium containing ampicillin (2 test tubes per person). Cultivate overnight at 37°C. After cultivation, store the bacterial liquid at 4°C. The samples will be used for the plasmid isolation experiment.

2.2 Plasmid DNA Isolation

In this experiment, we will perform isolation of plasmid DNA from the E. coli cells using the alkaline SDS method. In alkaline conditions, the denaturation of DNA (dissociation into single strands) becomes easier to achieve. However, the dissociation of closed-ring plasmid DNA is not completed. When the solution is returned to neutral pH, DNA reassembles randomly and precipitates from the solution. However, plasmid DNA that did not dissociate completely returns to the previous CCC state and remains dissolved in the solution. Using this method, it is possible to separate chromosomal DNA and plasmid DNA and then isolate the CCC state plasmid by further alkali processing method. In this experiment, we will isolate the plasmid DNA from the 2 liquid cultures of E. coli using the alkaline SDS method.

Equipment

• Micropipettes

• Micropipette tips

• Tubes

• Centrifuge

• Vortex mixer

• Incubator

Reagents

• Culture solution of E. coli NEB 10-beta strain retaining pUC19

• Solution I (Sol I): 50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA(Ethylenediaminetetraacetic acid), pH 8.0

• Solution II: 0.2 N NaOH, 1% (w/v) SDS

• Solution III: 3 M potassium acetate, 11.5% (v/v) acetic acid

Precautions

Do not discard the bacterial solution and bacterial waste in the sink after the experiment. Place the microtubes and micropipette tips in the waste bottle on the laboratory bench and discard in an autoclave bag at the laboratory entrance as appropriate. Dispose of the bacterial cell suspension liquid and bacterial cell culture waste into a dedicated cell waste container provided in the laboratory.

NaOH and phenol/chloroform denature protein, which affects human epidermal tissues and cells. If one of those solutions gets on your hands, wash it off with plenty of running water. If it enters your eyes, it may cause blindness, so wear safety glasses. There may be people handling these reagents around you, so act with due consideration and awareness.

Sample storage

After performing the experimental procedure, place the 2 tubes containing isolated plasmid DNA upside down on a Kimwipe, removing the alcohol by allowing it to volatilize. Store the samples at -20°C and use them for the next experiment (DNA Cutting Using Restriction Enzymes).

Procedure

1) Collection of bacteria

Collect 1.0 ml culture broth into two microtubes. Centrifuge at 12,000 rpm for 60 seconds (at 4°C). Remove the medium using 1,000-µl pipette and discard it into a beaker. Keep the tip as far away from the bacterial pellet as possible as the fluid is withdrawn from the tube.

2) Bacteriolysis and alkaline denaturation

Resuspend the bacterial pellet in 100 µl of Solution I by vigorous mixing with vortex mixer. It is essential to ensure that the bacterial pellet is completely dispersed in Solution I. Add 200 µl of Solution II. Close the tube tightly, and mix by inverting the tube upside down ten times. Make sure that the entire surface of the tube comes in contact with Solution II. Do not vortex. Store the tube on ice to prepare for neutralization heat generated in the next step.

3) Neutralization

Add 150 µl of ice-cold Solution III. Close the tube tightly, and mix by inverting the tube upside down for 20 seconds to disperse Solution III through the viscous bacterial lysate. Store the tube on ice for 2 minutes. Centrifuge at 12,000 rpm for 5 minutes (at 4°C) in a microfuge. Transfer all of the supernatant to a new tube, taking care not to take the white precipitate

4) Phenol/chloroform extraction

Add 450 µl of phenol/chloroform. Close the tube tightly, and mix vigorously with vortex mixer for 10-20 seconds. After centrifuging at 12,000 rpm for 3 minutes, transfer 200 µl of aqueous (uppermost) phase to a new tube, taking care not to take the inter and organic phase.

5) Ethanol precipitation

Add 400 µl of 100 % ethanol to precipitate DNA (and RNA), and mix vigorously with vortex mixer for 5-10 seconds. Centrifuge at 12,000 rpm for 5 minutes (at 4°C).

6) DNA washing

Remove the supernatant using 1,000 µl-pipette and discard it into beaker. Add 800 µl of 70% ethanol to wash the pellet. Detach the pellet from the tube wall with pipetting, and mix vigorously with vortex mixer for 5-10 seconds. After centrifugation at 12,000 rpm for 5 minutes (at 4°C), remove the supernatant completely with 1,000-µl pipette. Put the tubes upside down on a Kimwipe and dry the pellets.

2.3 DNA Cleavage Using Restriction Enzymes

Restriction enzymes are indispensable in gene disruption and gene expression, which are often carried out in molecular biology and gene manipulation research. Restriction enzyme is a type of endonuclease, and is an enzyme that recognizes and cleaves a double-stranded DNA at a specific base pair sequence. Using this enzyme, the DNA is cut and the ring shaped plasmid DNA attains straight chain structure. The restriction enzymes handled in this experiment are HindIII and PvuII. HindIII is a popular restriction enzyme, whose recognition sequence is contained inside MCS (multiple cloning site) of plasmid DNA. It cleaves the plasmid at 1020 bp. PvuII has recognition sequences at two positions: 841 bp and 1163 bp, both outside of MCS. The DNA fragment generated when pUC19 is cleaved with HindIII is around 2.7 kb long. In case of cleavage using PvuII, around 2.4 kb and 0.3 kb long fragments are formed.

Equipment

• Micropipettes

• Micropipette tips

• Tubes

• Centrifuge

• Incubator

Reagents

• Isolated plasmid DNA (2 tubes)

• TE

• TE-RNase

• Restriction enzyme HindIII-HF

• Restriction enzyme PvuII-HF

Remarks

RNA digestion and restriction enzyme digestion are carried out by individuals.

Procedure

1) RNase digestion

Label the 2 microtubes with DNA samples: write TA on one, TA-RNase on another together with your name. Add 15 μl of TE/ TE-RNase to the microtubes and tap several times to dissolve the pellet. Collect the solution at the bottom of the microtubes by flash (centrifuge for about 10 s). Keep at 37°C for around 1 hour.

2) Restriction enzyme digestion

We will prepare the following samples for the electrophoresis that will be carried out in the next experiment (Agarose Gel Electrophoresis):

Sample 1) No treatment with restriction enzyme; DNA sample dissolved in TE

Sample 2) No treatment with restriction enzyme; DNA sample dissolved in TE-RNase

Sample 3) Treated with HindIII-HF; DNA sample dissolved in TE-RNase

Sample 4) Treated with PvuII-HF; DNA sample dissolved in TE-RNase

Prepare four 1.5 ml microtubes and attach the seals to all of them. Write your name and solution type on each (empty) tube. Prepare the solutions by referring to Table 1.

Sterile distilled water and NEB buffer are present in all samples, and the amount of liquid is also the same. In this case, in order to save time and effort, it is better to create and use a premix for the number of samples plus one: sterilized distilled water 12 μl × 5 = 60 μl + NEB buffer 2 μl × 5 = 10 μl. The prepared premix is added to each tube (12 + 2 = 14 μl per tube).

After adding 14 µl of premix to each tube, keep adding the remaining solutions in the same order as in Table 1. Add the restrictive enzymes last. Replace the micropipette tip between adding different solutions.

After flipping the microtubes lightly, flash and let the reaction proceed at 37ºC for one hour. Avoid violent mixing with vortex mixer etc.. While the reaction proceeds, move on to the next experiment.

2.4 Agarose Gel Electrophoresis

In this experiment, we will separate the linear double-stranded DNA by agarose gel electrophoresis, and the mobility of the DNA fragments will reveal the size (in base pairs) of the specific fragments. Separation in which the molecular size of nucleic acids, proteins, etc. can be determined is extremely important in genetic engineering and molecular biology research. Agarose gel electrophoresis is one of the most popular of the techniques for detecting nucleic acids and determining their size.

Equipment

• Microwave

• Electrophoresis tank

• Transilluminator

• CCD camera

• Thermal printer

Reagents

• TAE buffer

• Agarose

• DNA marker (1 kb DNA ladder, NEW ENGLAND BioLabs)

• 6x DNA electrophoresis dye (orange G + xylene cyanol)

Procedure

1) Preparation of 0.8% agarose gel

Agarose gel should be prepared by a group of three to four people. Weigh 0.32 g of agarose into a 100 ml conical flask and add 40 ml of 1× TAE. Cover with a plastic wrap and heat in a microwave oven to completely dissolve the agarose. Use heat-resistant gloves when holding a flask to avoid burns. After dissolving agarose, leave the liquid to cool for 15 min in order to avoid the deformation of gel rack.

2) Solidification of agarose gel

Place two small gel cassettes on the gel rack and pour about 20 ml of the cooled agarose solution in each of them. If there are air bubbles on the surface, remove them with a pipette tip. Set a 12-hole comb on 2 gel plates and wait about 20-30 minutes for the gel to solidify completely. Wash the conical flask quickly so that the gel does not solidify inside it. Cover the gel with plastic wrap to avoid contamination with dust etc..

3) Electrophoresis of DNA samples

By applying high concentration glycerine etc. to a DNA sample used in electrophoresis, we make its specific gravity and viscosity higher than that of water. Therefore, we can prevent the DNA sample that we inject into the well from diffusing in the electrophoresis tank.

Add 3 μl of 6 × electrophoresis dye to the DNA sample (samples 1 to 4) and mix by pipetting or tapping. Carefully pull the comb out of the gel. Apply 6 μl of the DNA marker and 8 μl of each of the 4 samples into 5 wells.

4) Electrophoresis

Carefully place the gel in the electrophoresis tank so that the sample does not diffuse from the wells. Put the acrylic safety cover (lid) on the electrophoresis tank and start the migration at the voltage of 100 V. The migration is finished when the Orange G dye (flowing faster than Xylene cyanol) moves to the lower edge of the gel.

5) DNA staining and detection

After electrophoresis, put on gloves, transfer the gels to the vat (1 gel / 1 vat), and put a tape with your name on it. Take the bat to a room with an imaging device, add a fluorescent reagent for nucleic acid detection, and stain for 10 minutes. When the fluorescent reagent for nucleic acid detection binds to DNA, the fluorescence due to ultraviolet light or light of a specific wavelength is increased, and it is possible to fluorescently detect trace amounts of DNA (several ng or more). Discard the migration buffer of the electrophoresis apparatus while staining proceeds. Put the gel in a trans-illuminator, making sure there are no air bubbles, close the cover, turn on the lamp, check the DNA band, and take a picture.

6) The relationship between the size of the DNA molecule and its mobility

Plot the mobility of the DNA fragments (x axis) against the base pair (y axis, logarithmic scale) for the DNA marker sample. In the case of an unknown DNA, it can be electrophoresed side by side with a standard fragment (DNA marker) which has a known base number, so its size can be estimated if it falls within the range of the marker. The target DNA fragment marker has a different nucleotide sequence, but the molecular structures are basically the same. Thus, although the bases are different, comparison is possible because there is almost no difference in molecular size and charge state.

Sample 3 (Treated with HindIII-HF; DNA sample dissolved in TE-RNase):

The mobility for lane 4 was 100.0 mm.

DNA fragment size = 29210e^{-0.023×mobility} bp = 29210e^{-0.023×100.0} bp = 2928.56 bp ≈ 2.92 kb

The literature value for the DNA fragment size when pUC19 plasmid DNA was cut with HindIII is around 2.7 kb. Therefore, relative error is 8.1%.

Sample 4 (Treated with PvuII-HF; DNA sample dissolved in TE-RNase)

The mobility for lane 3 was 104.3 mm.

DNA fragment size = 29210e^{-0.023×104.3} bp = 2652.79 bp ≈ 2.65 kb

The literature value for the DNA fragment size when pUC19 plasmid DNA was cut with PvuII is around 2.4 kb. Therefore, relative error is 10.4%.

However, PvuII cuts the plasmid DNA at two sites, so we expect 2 fragments to be visible in the migration pattern. Apart from the 2.4 kb line, there should be another line at 0.3 kb. The absence of the smaller fragment line on the spectrum may be caused by insufficient concentration of DNA in the sample.

However, we can clearly see that the fragment resulting from HindIII action, corresponding to one cleavage site, is longer than the fragment resulting from PvuII action. This suggests that PvuII cut the plasmid DNA at more than one site, creating 2 DNA fragments, even though the smaller fragment is not visible.

To obtain better results and a migration pattern that would show the smaller DNA fragment for sample 4, we should ensure that the DNA concentration is higher than in the current experiment.

The possible source of error that increased the value of relative error for samples 3 and 4 is the DNA marker used. As we can see in Figure 2, many of the points do not line on the line of best fit. Reducing random error in the electrophoresis of Marker DNA could yield more accurate values of DNA fragment sizes.

Moreover, in order to be able to use my own samples’ results (instead of teacher’s) in the experiment, it is crucial to ensure that none of the reagents and dyes are degraded. It is advisable to check the expiration date of the solutions, and to confirm that they were stored in the conditions that would prevent deterioration of their quality.

In case of sample 2 (no treatment with restriction enzyme; DNA sample dissolved in TE-RNase), we can see only one band. It is likely that that band is caused by the presence of plasmid DNA in CCC state. The reason why it is possible to CCC state band to appear in the migration pattern is the fact that sample 2 was not treated with any kind of restriction enzyme that would cut the ring shaped plasmid DNA, so the structure remains intact.

Looking at the spectrum of sample 1 (no treatment with restriction enzyme; DNA sample dissolved in TE) in Figure 2, we can see a line at the same position as in sample 2. However, the line is around 2 times as wide, and much brighter than in case of sample 2. Moreover, we can see a bright region, probably composed of many bands on top of each other. As the migration of that region is higher than that of the main bright band, we can deduce that the fragments are small. The reason why the migration pattern of sample 1 possesses that multiple fragment region is the absence of RNase in the solution of sample 1. RNase is an enzyme that cuts RNA into small pieces. Those small fragments are able to travel through agarose gel much quicker than the uncut RNA. Therefore, as we can see in the pattern of sample 2, which contains RNase, the small fragments are “washed off” the electrophoresis plane in question. This demonstrates the usefulness of RNase digestion (breaking down RNA into smaller fragments) in clearing the electrophoresis migration spectrum, which ensures that we investigate only the DNA fragments of interest and that we can see their bands clearly, without “noise”.

The bands corresponding to CCC state appear clearly only in sample 1 and 2, but we can also see a “ghost” band at the same position in sample 3 and 4. This could mean that restrictive enzymes were not able to cut through all of the plasmid DNA, and a small quantity of it remained in the CCC state.

Theory

Which plot of the marker (linear or logarithmic) data is better? What does this tell you about the migration of the DNA fragments through the agarose gel?

We plot the marker data on a logarithmic scale, not linear. This is because the migration (distance traveled in gel) of the DNA fragments is inversely proportional to the log of the DNA fragment size or molecular weight, so logarithmic scale is simply more suitable- the data is easier to see and analyze this way, as we get a straight line like in Figure 2 above. While you could technically plot your data on linear scale, you would get a graph with a very steep slope, so the data readability would be pretty poor. Thus, it is not recommended.

The mechanism of antibiotic resistance

pUC19 used in this experiment has incorporated the bla gene, which confers a characteristic of ampicillin resistance. But how exactly does the protein encoded by the bla gene make the bacteria resistant to this antibiotic?

In order to form, maintain and remodel its cell wall, bacteria need to continuously cross-link the peptidoglycan chains, which are one of the main components of the bacterial wall. The enzyme cross-linking the peptidoglycans is called DD-transpeptidase.

One of the most popular antibiotics used nowadays to fight the bacteria come from the beta-lactam family. These antibiotics are called beta-lactams because they incorporate a beta-lactam ring in their structure. Beta-lactam ring binds to DD-transpeptidase, preventing it from cross-linking the peptidoglycans, thus inhibiting further wall formation. With a weakened cell wall, bacteria is unable to survive, giving in to the outside water/molecular pressure.

However, bacteria can become immune to beta-lactams. In this experiment, we incorporated pUC19 plasmid DNA into the genome of E.coli, making it resistant to ampicillin. pUC19 contains the bla gene, which encodes beta-lactamases. Beta-lactamases have the ability to hydrolyze the beta-lactam bond of the beta-lactam antibiotics. As the result, beta-lactams lose their ability to prevent the E. coli cell wall from forming. Beta-lactams include penicillins, cephalosporins etc., and ampicillin is a penicillin derivative, so it is also rendered ineffective by the beta-lactamases produced by E. coli thanks to bla gene in pUC19 plasmid.

Why is chromosomal DNA is agglutinated and only plasmid can be recovered if they are both rapidly neutralized after alkaline denaturation by alkaline miniprep method?

Miniprep method refers to preparation of plasmid DNA from a small number of cells, usually cultured over a short period of time. After denaturation with an alkaline solution, such as SDS used in this experiment, the DNA of the cell becomes single-stranded. When the solution is rapidly neutralized, for example with potassium acetate, chromosomal DNA, which has a large, complex structure, will not be capable of renaturation. Instead it will form short regions of double-stranded chain where the nucleotide sequence is similar, which will result in aggregation. Chromosomal DNA will be agglutinated and become insoluble, thus precipitating from the solution. However, this is not the case for plasmids. Plasmids are small and have a circular shape, so even after denaturation the strands do not end up far away from each other. Thus, when the solution is neutralized, plasmids (that are not as large and complex as chromosomal DNA), are able to return to the double stranded state (renature) easily, and thus they remain dissolved in the solution. Then, plasmids can be easily recovered by removing the precipitated agglutinated chromosomal DNA (for example centrifuge so that the agglutinated DNA forms a pellet and recover the supernatant containing only the plasmids).

Another plasmid example

In this experiment, we handled pUC19 plasmid, but there is a plethora or incredibly useful plasmids used in genetic engineering. Here, I would like to introduce a plasmid which can be used for in-vivo imaging.

pEF.myc.ER-E2-Crimson is a plasmid that contains the gene encoding the fluorescent protein called E2-Crimson. It is used for in-vivo visualisation of Endoplasmic Reticulum. The hosts for the plasmid are animals, and the protein itself was first derived from Discosoma sp..

Since E2-Crimson has low cytotoxicity in bacterial and mammalian cells, it can be suitable for live-animal imaging. Moreover, it has a short maturation time (26 min half-time for chromophore maturation at 37 °C) and excitation and emission in far-red region, which makes it even more desirable, since in living tissues absorption and light scattering at wavelengths larger than 600 nm are quite low. In addition, E2-Crimson has particularly high brightness and stable pH, pKa being equal to 4.5.

As the result, it is a good candidate for being used in whole-cell labelling of living animal cells, also in sensitive cells such as stem cells and primary cells[9]. It can also find application in multicolor labeling experiments, when used together with green and orange fluorescent proteins.

Bibliography

[1] Wageningen University Bioinformatics. 2019. Miniprepping. http://www.bioinformatics.nl/molbi/SimpleCloningLab/miniprepping.htm. [Accessed 28 June 2019].

[2] Susan Chow. 2018. Penicillin Mechanism. https://www.news-medical.net/health/Penicillin-Mechanism.aspx. [Accessed 28 June 2019].

[3] PubChem. 2016. Bla - beta-lactamase (Escherichia coli). https://pubchem.ncbi.nlm.nih.gov/gene/bla/Escherichia_coli. [Accessed 28 June 2019].

[4] Drug Bank. 2005. Ampicillin. https://www.drugbank.ca/drugs/DB00415. [Accessed 28 June 2019].

[5] Add Gene. 2019. pEF.myc.ER-E2-Crimson. http://www.addgene.org/38770/. [Accessed 28 June 2019].

[6] FPbase. 2019. E2-Crimson. https://www.fpbase.org/protein/e2-crimson/. [Accessed 28 June 2019].

[7] Rita L. Strack, Birka Hein, Dibyendu Bhattacharyya, Stefan W. Hell, Robert J. Keenan, and Benjamin S. Glick. 2009. A Rapidly Maturing Far-Red Derivative of DsRed-Express2 for Whole-Cell Labeling

Biochemistry 2009 48 (35), 8279-8281. DOI: 10.1021/bi900870u. https://pubs.acs.org/doi/10.1021/bi900870u. [Accessed 28 June 2019].

[8] Takara Bio. 2019. E2-Crimson fluorescent protein. https://www.takarabio.com/products/gene-function/fluorescent-proteins/fluorescent-protein-plasmids/far-red-fluorescent-proteins/e2-crimson-fluorescent-protein. [Accessed 28 June 2019].

[9] Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of visualized experiments : JoVE, (62), 3923. https://doi.org/10.3791/3923