Advantage of Expression cloning in pET28a (+) vector

Gene synthesis & pET28a (+) cloning:

Eurofins Genomics India now offers expression cloning in the pET28a (+) vector at its Bangalore facility. This service includes gene synthesis for low to mildly complex genes, opening many opportunities for protein expression projects. Our in-house GENEius software optimizes parental sequences for various expression systems, including E. coli, mammalian ells, insect cells, and yeast (S. cerevisiae and P. pastoris).

Our oligo manufacturing, verified by MALDI-TOF QC, ensures zero mutations. All genes are produced under GMP, GLP, and ISO 9001:2015 compliance. Each gene is carefully curated and checked by dual-restriction digestion and Sanger sequencing, ensuring 100% mutation-free genes. Additionally, we offer peer-to-peer interaction with our technical staff for project customization.

Why do we select the pET vector for expression cloning?

• The pET (Plasmid for Expression by T7 RNA Polymerase) system is renowned for producing large amounts of protein. Over 220,000 research articles have utilized this system, with more than 40,000 involving pET28a (+). Key advantages include:

  • Strong T7 Promoter: Increases protein production.
  • Common Restriction Sites: Especially upstream of the T7 promoter, making it ideal for expression cloning.
  • Lac Operator Regulation: Suppresses uninduced expression.
  • Self Lac Repressor Encoding: Reduces promoter leakiness, especially beneficial for bacteria with lac repressor deficiencies.
  • Medium Copy Number (20-25 per cell): Allows high expression levels without overloading the cell.

Process and Quality validation:

Step 1: Gene Synthesis and cloning in Eurofins Standard pEX series vectors.

1. Gene Assembly: To reduce the complexity of tested genes (1.6 kb), the gene design program helps to create multiple fragments (3 fragments in this case study). 

Fig 1:  With our effective gene assembly program, 3 fragments were assembled to produce double-stranded gene fragments.

Step 2: Reassembling of full-length gene and cloning in Eurofins Standard vector (pEX series):

Fig 2: Further, the full-length gene was reassembled in our Eurofins Standard pEX series of vector followed by the confirmation by RD assay and Sanger sequencing. (A) Eurofins Standard vector for initial Cloning of your 100% mutation-free gene, (B) Re Assay for the confirmation of Gene Insert in PEX vector.

Step 3: Sub-cloning of GOI to pET28a (+) expression vector:

After all the confirmations, the full-length insert was subjected to the sub-cloning in the pET28a (+) expression vector.

Fig 3: Sub-cloning of the gene of interest to the pET28a (+) vector. (A) Screening of GOI subcloned in pET 28a (+) vector, (B) Commercial pET28a (+) vector Map (Novagen).

Step D: RE assay and Sangar sequencing confirmation:

To confirm the desired gene construct, various colonies were selected for further subjected to RE digestion and Sanger sequencing.

Step 4 : Restriction Digestion (RD) assay and Sanger sequencing confirmation:

To confirm the desired gene construct cloned in pET28a (+), multiple colonies were selected and further subjected to RD assay and Sanger sequencing.

Fig 4: RD assay and Sanger Sequencing for final confirmation of GOI sub-cloned in pET28a  (+). (A) RE confirmation of GOI Subcloned in pET28a(+) vector, (B) Sanger Sequencing confirmation of GOI Subcloned in pET28a(+) vector.

Goals

1. What are the expected project outputs? Each gene is well-curated with a unique gene design program. The synthesized dsDNA will be packed in a pET28a (+) vector and delivered to the client after all the requisite test confirmations.
2. What information is shared with customers? All the stepwise information will be shared with the client from the assembly of the gene to the packaging supported by Sanger Sequencing electronic data.

USP of the product

• In-house GENEius codon optimization software.
• Ease of customization with the pET28a (+) vector.
• Fastest TAT (8-10 Working Days).
• Peer-to-peer interaction with the client for the project customization.
• QC Doc, Gel Image, restriction digestion profile, and Sanger sequencing electronic data will be shared.
• Ease of opting mini (1-2 ug), midi (>4ug-8ug), and maxi prep (>10ug-12ug) as per the demand.

Place your orders and inquiries at  https://www.eurofins.in/genomics/enquire-now/

Enquire Now