Bacteriophage lambda has been used as a host vector for generation of genomic and cDNA libraries for quite some time; efficient libraries are generated primarily because of the phage particles ability to efficiency infect the bacteria
in vitro. Some research workers prefer to use the original lambda as a vector while others, including myself, like to propagate the gene fragments in a cosmid vector. Promega’s Packagene® Lambda DNA Packaging System was the ultimate choice when I was looking for a cost-effective and productive system for generating a gene library of my bug in a cosmid vector. In the past, I faced lots of problems getting the desired number of clones. After using Promega’s Packagene® Lambda DNA Packaging system, my whole lab asks me for help when they face similar problems.
Promega’s Packagene® Lambda DNA Packaging system is designed for both cDNA and genomic library construction. It is derived from a unique one host system and is easy to use as it contains all the components and buffers needed to perform the packaging reaction in a single tube. One just has to add the ligation reaction to it. The packaging extracts have been produced from an E.coli C bacterial host strain; the strain lacks the EcoK restriction system, so the libraries are not biased by the K-12 restriction system of the host. This can also be evaluated by using the lambda c1857 Sam7 DNA as a sample without depleting the sample DNA. One may suspect the packaging efficiencies to be low with such a single strain system, but in my practice I have found better results than any other system.
Originally, Promega had not designed the extracts for propagation of a cosmid library, and if one goes through the protocol provided with the product, it never mentions the use of a cosmid vector anywhere. But because of many failures in the past with other systems, I decided to give it a try because the basic principle for a cosmid or a lambda vector relies on the presence of cos sites in the vector. During the ligation reaction, long concatamers are formed which are recognized by the packaging proteins and a certain length of DNA is packaged into the phage head. This size selection is a big help because it reduces the total number of required clones in a given library. With cosmids, after packaging and transfection, the cosmid along with the insert goes into the bacterium and gets circularized in the form of a plasmid which is then very easy to manipulate in downstream processes.
As the original protocol was not designed for the propagation of cosmid clones, I recommend a few modification when using a cosmid vector along with the Packagene® Lambda DNA Packaging system. For preparation of bacterial culture stock and infection, I recommend you strictly follow procedure #2 as stated in the protocol provided by Promega. I do not use overnight cultures because they often reduce the titers after infection because dead cells may be there which may bind to recombinant phages and decrease the successful infection rate. There is a sample ligation reaction protocol in the manual which can easily be manipulated for one’s own purposes. I recommend a 4-6 times higher insert to vector ratio for the packaging reaction. Use of PEG (normally added in quick ligation kits) is not recommended as it can promote blunt-ended fragments by macromolecular crowding. In addition, PEG is inhibitory to the packaging reactions.
The thing I like most about this system is the volume of the packaging reaction. It is a large-scale reaction (50 µl) which can easily package 0.5 µg to 5 µg in a volume of 5-10 µl. Normally, with other packaging systems, one has to use a diluted ligation reaction, and the result is few clones. I recommend using the maximum amount of DNA for ligation so there are more chances of getting recombinant clones at the end. The packaging step in the protocol is quite easy to follow and I recommend that you stick to it.
For titration of phage libraries, the protocol is excellent, but for titering cosmid clones, it needs some modification. This is not as complicated of a procedure as it would seem to be. Make dilutions in the similar manner as stated in the protocol and spread 50 µl - 100 µl on a LB plate with the proper antibiotic. The rest of the calculations are the same.
I have used this system many times and have obtained successful results. With optimized ligation conditions, which one would have to evaluate by oneself, this system is quite successful in producing recombinant phages whether to maintain as phage libraries or as cosmid clones. The host strain provided is E.coli LE392 which lacks the E.coli K restriction system, but is rec+. It is a permissive host, allowing both recombinant and parental phage to grow. It does not allow color selection of recombinants or IPTG induction of expression. Also, since LE392 contains lon protease activity, fusion proteins are often less stable in this host. In addition, other strains like E.coli HB101 and VCS257 can also be used efficiently with this system. When using LE392 strain, I recommend (if color selection is not required) that LE392 be the primary strain for amplification of recombinant phage and for screening a cDNA library with nucleic acid probes.
In conclusion, Promega’s Packagene® Lambda DNA Packaging system is very useful and convenient for constructing cosmid or phage libraries despite the fact that it is primarily marketed for propagating phage libraries only.