Advantage of Expression cloning in pET28a (+) vector

Gene synthesis & pET28a (+) cloning:

Eurofins Genomics India has recently added expression cloning in the pET28a (+) vector followed by the gene synthesis of low to mild complex genes at its facility in Bangalore. This opens many opportunities for building multiple protein expression projects to leverage the diversification of in silico gene synthesis to be exploited for protein expression studies. Our in-house GENEius software ensures that requested parental sequences are best optimized with the choice of a heterologous organism to enhance the protein expression in E.coli as well as other expression systems including mammalian cells, insect cells, and yeast (S. cerevisiae and P. pastoris).  

Our in-house oligo manufacturing enabled by the MALDI-TOF QC ensures ZERO-MUTATION. All the genes are manufactured under GMP, GLP, and ISO 9001:2015 compliance. Each base of the gene is well-curated by the gene design program and every clone is further checked by dual-restriction digestion as well as Sanger sequencing. This ensures that you get a 100% mutation-free gene.  

The biggest advantage is peer-to-peer interaction with our Eurofins technical staff regarding the customization of a project. You can call us for “CUSTOMIZATION”  of gene.  

Why do we select the pET vector for expression cloning?

The pET (Plasmid for expression by T7 RNA polymerase) expression system is a well-recognized system that allows the production of a large amount of a desired protein. So far, >220,000 research articles have been published, wherein >40,000 articles have been published in expression cloning in pET28a (+). There are several key advantages of having the expression cloning in a pET vector:

• Having a strong known T7 promoter helps to increase protein production.
• Having many common restriction sites, especially upstream of the T7 promoter makes them the choice of vector for the expression cloning.
• A strong T7 promoter is regulated by the lac operator to suppress uninduced expression.
• The plasmids encode their own lac repressor. The major advantage of the pET selection, wherein few bacteria have lac repressor deficiency, this system further helps to reduce the leakiness of the promoter.
• Having a medium copy number (~20-25 per cell), allows for the high expression level of the T7 promoter without overloading the cell with many copies of the plasmid in addition.
  
  

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/

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