I am trying to clone a large (11kb) and toxic gene in an expression vector. Therefore, I am using the EPI400 CopyCutter strain that is maintaining the plasmid at a low copy number to allow bacteria growth. Attached is the protocol for this strain.
I have to grow the bacteria on plate at 28 C for 2 to 3 days to be able to get colonies.
I did a colony PCR and got 20 positive colonies.
When I put them in liquid culture (5mL to 10mL), some colonies never grown, even at 28 C. Some of them did grow (2 days incubation 28C, 250rpm in aeration culture tube, OD600 above 2.0), but then I only get a really low yield after induction of the high copy production in 100mL culture in flasks.
I tried this several times but still got a really low yield (10ng/uL, maximum 15).
I used this strain for other toxic genes and was able to get more than 40ng/uL (which is around what I need to be able to sequence my plasmid and verify the correct insertion of my gene).
I am using this kit for plasmid extraction Wizard® Plus SV Minipreps DNA Purification Systems
Any suggestions on how I could improve my plasmid yield?
Any suggestions to be able to grow the bacteria in liquid culture after getting them selected on the plate?
Thank you.
To optimize plasmid DNA yield when working with toxic genes:
By implementing these strategies, you can improve plasmid DNA yield while working with toxic genes, such as when using CopyCutter EPI400. Experimentation and optimization may be necessary to find the best approach for your specific gene and experimental setup.
You will probably have to switch to the gene expressed under a tightly controlled promoter like PBAD (arabinose) or the tetracycline inducible (or anhydrotetracycline inducible) promoter. The tet repression system is pretty amazing, I've used it to clone the tse2 gene from P. aeruginosa on a plasmid, which causes growth arrest in E. coli if any of it is expressed at all.
You could also consider switching to another bacterial species entirely, depending on what your end goal is and what the gene is. I had to switch from cloning in E. coli to L. lactis due to a similar scenario - when cloning a gene from E. faecalis under a constitutive promoter into a shuttle vector in E. coli, most colonies did not grow on subculture and a few grew at 30° after 48 hours. When I sent the ones that grew for sequencing literally every clone had a transposon insertion disrupting the gene. When I cloned the plasmid in L. lactis, there was no issue.
There's unfortunately a difference in how you deal with cloning genes that are "toxic" when they're on a high copy number plasmid and genes that are capital T Toxic.
To enhance your plasmid DNA yield with CopyCutter EPI400 for a toxic gene, consider these steps:
1. **Optimize Culture Conditions**: Adjust culture conditions such as temperature, pH, aeration, and medium composition to maximize plasmid yield while minimizing toxicity.
2. **Induction Timing**: Determine the optimal induction timing to balance gene expression and cell viability. Inducing the toxic gene too early or too late can reduce plasmid yield.
3. **Media Supplementation**: Supplement the growth medium with additives like glucose, lactose, or IPTG to regulate gene expression and alleviate toxicity.
4. **Temperature Control**: Optimize the incubation temperature to maintain cell viability while promoting plasmid replication and stability.
5. **Use of Host Strains**: Utilize host strains specifically designed for toxic gene expression, such as those with inducible promoters or genetic modifications to enhance plasmid stability.
6. **Scale-Up and Optimization**: Scale up your culture volume while continuously optimizing conditions to achieve higher plasmid yields without compromising cell health.
7. **Purification Method**: Select an appropriate plasmid purification method that effectively removes cellular debris and contaminants while preserving DNA integrity.
8. **Consider Alternative Systems**: If toxicity remains a challenge, explore alternative expression systems or vectors that are better suited for toxic gene expression.
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