Hello fellow researchers,

I have been attempting to construct a fusion protein plasmid for a while but have yet succeeded. Any suggestions beyond this point would be highly appreciated.

Essentially, the goal is to insert a sequence into a plasmid that we obtained from Addgene. Since this is a fusion construct I thought it would be desired to delete the stop codon from the original sequence, followed by the insert, and stop codon in the end. The vector that I am using is pVITRO-Hyg from Invivogen.

Process:

We have successfully extracted the vector and insert plasmids and verified their sequence with Sanger sequencing (we did not sequence the full constructs but a partial sequence of >900 consecutive reads). Then I linearized the vector (8.6 kb) with Phusion high-fidelity polymerase end-to-end but deleted the stop codon in the sequence where I wanted to have the insert in. And also designed 15-20 overlapping nucleotides on the insert primers so I can assemble them with Gibson cloning using the NEB HiFi assembly kit. After DpnI-digesting both sequences for 2 hours at 37C and purifying, I mixed the DNA 1:2 according to the NEB kit, and incubated it with the assembly reagent for 1 hour at 50C. The assembly was confirmed by PCR primers annealing to the vector sequence that amplify across the vector-insert junction. In the agarose gel I obtained ~90% band showing vector + insert, 8% unreacted vector alone, and 2% unspecific amplicons.

(* possibly due to a primer issue, I always get a nonspecific band in the linearized vector, I either ignored it since I have the correct sequence amplified, or I used gel extraction to extract the correct sequence).

Then I purified the reaction product again and obtained ~60 ng/uL DNA overall, I used ~10 ng to transform 25 uL competent NEB 10-beta cells using a standard heat shock protocol (thaw, heating at 42C for 30s, chill on ice for 5 min, add 475 uL NEB 10-beta SOC and shaking at 37C for 1 hour before inoculating 100 uL on an agar plate with 75 ug/mL Hygromycin B).

Result:

The cell growth was slow, and I was able to get decently sized colonies after ~24 hours of growth. I was thinking this is due to a mix of slow-growing 10-beta cells and a longer lag phase for Hygromycin resistance. > 50 colonies grew, a few colonies also grew with mock-transformed cells (without DNA added, serving as negative control). When I grew the colonies in liquid LB with 100 ug/mL hygromycin, the growth was also slow. I extracted the plasmid using Promega Wizard Plus SV Minipreps. The yield was relatively low around 60-100 ng/uL for a 10 mL extraction, likely due to the slow growth of cells. And when I run those on a gel, I only get a single band that is above the 1 kb NEB DNA ladder (the plasmid vector shows 3 bands, including a linear band at the correct size). When I send the plasmid for sequencing, the primers for both insert and the vector did not anneal well, and for those that did (with poor priming), the sequence was vastly scrambled - it did not contain my vector or insert.

I have done this multiple times now and also did colony PCR. However, most of the colonies that grew after transformation did not contain either the vector or insert. For a few instances, I was able to use colony PCR to see the correct inserted sequence, but those colonies were not able to grow in liquid LB (interestingly those colonies are a few weeks old, while newly-grown colonies grew well in liquid LB but do not contain the right sequence). I also used restriction digestion of the extracted plasmid and ran a gel - there was no significant movement of the band before or after digestion.

I attached some imaged gels,

The first image is the correct amplification of the vector + plasmid (the second band, 1.7 kb), whereas the empty plasmid is shown with the first band (1 kb). This is a previous attempt and I have been able to increase the density of the second band to ~90% after designing a longer overlap sequence and gel extraction.

The second image is what I also get after extraction - bands above the 1 kb ladder that are resistant to restriction digestion and show a scrambled sequence upon sequencing.

Please let me know if you have suggestions, thank you!