Can you help me find a detailed method/protocol/paper that shows step-by-step walkthrough in checking the (1) plasmid copy number and (2) transformation efficiency in lactobacilli or at least Gram-positive bacteria? Thanks in advance!
For plasmid copy number:
1. Plasmid p256 from Lactobacillus plantarum represents a new type of replicon in lactic acid bacteria, and contains a toxin–antitoxin-like plasmid maintenance system.
2. Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli.
3. Calculation of Copy Number Standards for PCR Screening Assays
4. Digital mRNA profiling (Although it is designed for RNA it could easily be adapted for detecting plasmid DNA. It would be simple and fast)
For calculating Transformation efficiency:
1. http://www.bioruffo.net/protocols_bacteria_transformation_efficiency.php (Online calculator available)
2. Efficient transformation of Lactococcus lactis IL1403 and generation of knock-out mutants by homologous recombination
3. http://2010.igem.org/Team:WITS-South_Africa/Gram_positives
Use of real time qPCR is one of the efficient method to check PCN. You can go through the following papers http://www.sciencedirect.com/science/article/pii/S0003269700946682#
http://www.sciencedirect.com/science/article/pii/S0167701205002320#
http://link.springer.com/chapter/10.1007/b12440
Transformation efficiency (TE) = Colonies/µg/Dilution
Colonies = the number of colonies counted on the plate
µg = the amount of DNA transformed expressed in µg
Dilution = the total dilution of the DNA before plating
Thanks for you help! I will definitely look into these comments. Very helpful!
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