Nobel laureate George Smith achieved the fusion expression of foreign proteins with phage coat proteins by fusing the gene of the protein of interest with the coding gene on the surface of the phage. This facilitates the efficient screening of large libraries of proteins and peptides based on their sequences.
This technique relies on a DNA vector, i.e., a phage, that combines the characteristics of a phage and a plasmid. This allows infected E. coli competent cells to produce new phage particles containing the protein or peptide of interest.
The design of a phage display library can include millions or more DNA clones carrying the target sequence, with the DNA sequence constituting the protein/peptide being cloned primarily onto a phage particle vector. The resulting phage particle library is transformed into E. coli cells, and after infection with a helper phage, a phage display library is generated. This library is then subjected to affinity screening against a previously fixed target. This process can be repeated several times to select the peptide/protein that binds most specifically.
This screening process makes phage display technology particularly suitable for discovering highly specific antibodies. Compared to hybridoma technology, it has several advantages:
1. Coverage of a large number of clones
2. Screening for toxic and non-immunogenic antigens
3. Library can be created using any animal (including humans)
4. Direct sequence availability, making further antibody design easier.
Phage display library can generate billions of different peptides and proteins. This requires highly efficient competent cells. Higher transformation efficiency can significantly increase the absolute number of recombinants and significantly reduce the cost of producing and screening peptide and protein phage display libraries. Competent cells optimized for DNA transformation can increase transformation efficiency up to ten times that of existing methods.
Electrotransformation of competent cells can achieve high transformation efficiency and is therefore recommended for producing large, highly diverse libraries. When an electrical pulse is applied, the membrane of competent cells is highly permeable, allowing genetic material to easily penetrate.
In phage display library construction, transformation efficiency directly determines library size and diversity. Obtaining a stable and efficient transformation system has always been crucial for experimental success.
AlpVHHs has launched the groundbreaking TG1 Super Electrocompetent Cells (Code: P021), a specially processed, highly efficient competent cell designed for electrotransfer, achieving a transformation efficiency of up to 4x1010 cfu/µg DNA, providing strong support for phage library construction.
● Transformation efficiency up to 4 x 1010 cfu/µg DNA
● Easily meets the needs of large-capacity library construction
● Improves positive clone coverage
● Optimized storage buffer system
● Supports long-term storage at -80℃
● Avoids efficiency degradation issues
● Designed specifically for electroporation, matching mainstream equipment parameters
● Reduces arc risk and improves experimental success rate
● Standardized workflow design
● Suitable for both novice and experienced users
● Phage display library construction and screening
● Antibody epitope mapping
● Peptide ligand identification
● Protein-Protein interaction identification
● Directed evolution of proteins
● Find tumor antigens, candidates for therapeutic antibodies, enzyme inhibitors, receptor agonists, etc.
1. Receiving and Storage: Upon receiving the product, check that the packaging is intact and immediately place the competent cells in a -80°C ultra-low temperature freezer.
2. Pre-Experiment Preparation: Quickly remove the competent cells from -80°C and place them in an ice box, waiting 10-15 minutes for complete thawing. Pre-cool the electroporation cuvette and EP tubes on ice, and simultaneously preheat the 2*YT/SOB medium to 37°C (heat shock can further improve electroporation efficiency).
3. Adding DNA: Add the DNA to be transformed into 25µL of competent cells, gently mix, avoiding air bubbles.
4. Electroporation: Transfer the cell and DNA mixture to the pre-cooled electroporation cuvette, taking care to avoid air bubbles.
After wiping the outside of the electroporation cuvette clean, set the parameters according to the recommended electroporation conditions (Bio-Rad Gene Pulser XcellTM electroporation reference conditions: voltage 1800V, resistance 600Ω, capacitance 10µF, electroporation cuvette: 1mm) and perform electroporation.
5. Immediately after the electroporation pulse ends, add 975µL of preheated 2*YT/SOB medium.
Resuspend the cells by pipetting up and down, then transfer the cell-containing medium to a sterile centrifuge tube.
6. Culture: Place the culture tubes on a shaker and incubate at 37℃ and 180rpm for 1 hour to allow cells to recover growth and express the resistance gene.
7. Plating and Transformation Efficiency Calculation: After culture, serially dilute the transformed cells, and plate 100µL of each onto agarose plates containing the specific antibiotic.
Incubate overnight at 37℃, and calculate the transformation efficiency the next day.
Efficiency calculation formula (taking 100 single clones on a 10-2 plate as an example):
100 x 102 x 10 x 105 = 1 x 1010 cfu/µg
Note: 100 is the number of single clones, 10² is the dilution factor, 10 is the conversion of 100µL to 1mL, and 105 is the conversion of 10pg to µg.
Components: 2 YT Agar Plates (per L), 16g tryptone, 10g yeast extract, 5g NaCl, 15g agar
Add all components to deionized water and adjust pH to 7.0 with NaOH. Autoclave and then cool to 55 ℃.
1. Upon receiving the product, immediately place it in a -80℃ ultra-low temperature freezer to avoid temperature fluctuations. Repeated freeze-thaw cycles are strictly prohibited, as they will severely affect transformation efficiency.
2. It is recommended to purify the ligation reaction product using a DNA recovery kit before transformation.
3. DNA samples must be dissolved in ddH2O or TE buffer to avoid high-voltage arcing during electroporation caused by salt ions.
4. EP tubes and electroporation cuvettes must be placed on ice before use.
5. Competent cells must be thawed on ice for 10-15 minutes before use; do not heat them by hand or in a water bath.
6. The culture medium must be preheated to 37℃; otherwise, transformation efficiency will be affected.
7. It is recommended to use each tube of competent cells immediately; repeated freezing and reuse are not recommended.
8. The DNA volume should not exceed 10% of the competent cell volume (i.e., ≤ 2.5µL / 25µL competent cells).
9. After electroporation, cells can be thawed and plated in SOB, 2*YT, or other conventional culture media. 10. Different electroporation instrument models may vary slightly; electroporation parameters need to be optimized. It is recommended to conduct parameter pre-experiments before first use.
11. Avoid air bubbles when transferring the cell-DNA mixture into the electroporation cuvette. Air bubbles can cause arcing, severely reducing transformation efficiency and even damaging the equipment.
12. It is recommended to set up a positive control (e.g., 10 pg pUC19 plasmid) for each experiment to facilitate the assessment of competent cell activity.
13. Except for preheating the culture medium, all steps involving contact with competent cells should be performed on ice or at a low temperature whenever possible.
14. Cells are relatively fragile after electroporation. Avoid vigorous shaking before thawing and culturing; handle them gently.
15. When transformation efficiency is high, direct plating without dilution may result in overly dense colonies, affecting single-clone picking. Therefore, appropriate dilution is necessary before plating.
Number of single clones on a 10⁻³ plate | Conversion efficiency |
44 | 4.4x1010 cfu/µg |
46 | 4.6x1010 cfu/µg |
52 | 5.2x1010 cfu/µg |
The competent state efficiency reached 4x1010 cfu/µg, and this batch is qualified.
AlpVHHs is an innovative biotechnology company focused on nanobody (VHH) technology, phage display platform and yeast display platform, committed to providing high-quality nanobody and nanobody discovery technology services to scientists, in vitro diagnostic and drug development companies worldwide, with core strengths including:
● Focus on VHH and antibody engineering technologies
● Independently optimized competent cell preparation processes
● Providing complete library construction solutions
AlpVHHs utilizes a special process to provide super-competent cells for electroporation, as well as super-competent cells already overinfected with helper phage M13K07, eliminating the need for additional helper phage infection during library packaging.
Code | Description | Transformation Efficiency (cfu/µg pUC DNA) |
P021 | ≥4x1010 | |
P021-2 | ≥4x1010 | |
P022 | ≥4x1010 | |
P022-2 | ≥4x1010 | |
P023 | ≥4x1010 | |
P023-2 | ≥4x1010 |
According to the instructions, a 1mm electroporation cuvette should be used with 25µl of competent cells per reaction. If the reaction volume exceeds 50µl, a 2mm electroporation cuvette is recommended.
The maximum volume of DNA/Ligation product added is 1/10 of the volume of competent cells used. The maximum amount of DNA/Ligation product added depends on several factors, such as the salt ion and PEG content in the sample. For example, using the positive control pUC19 DNA (10 pg/μL) in the kit, a maximum of 20 pg is recommended.
The most common method is to calculate the number of positive clone colonies (cfu) obtained after transformation per microgram of DNA. Assuming 10 pg of pUC19 plasmid is used for transformation, the final volume after adding culture medium is 1 ml (at this point, the DNA concentration is 10 pg DNA/ml). Then, the plasmid is diluted 1:10 (the DNA concentration becomes 1 pg DNA/ml), and 100 μl (containing a total of 0.1 pg of DNA) is spread onto two parallel plates. If the final colony count is 200 (the average number of colonies on both plates), then the transformation efficiency is: 200 cfu/0.0001 ng = 2 × 109 cfu/μg
The core difference lies in whether it has been pre-infected with helper phage.
Regular TG1 (P021) requires additional infection with M13KO7 helper phage during use, while P021-2 is pre-infected with helper phage and can be used directly for library amplification.
Therefore, P021-2 has the following advantages:
● Reduces 1–2 experimental steps
● Reduces human error
● Saves approximately 1-2 days of experimental time
