pMECA is a cloning vector which contains 44 unique restriction sites, including 9 rare-cutters, all within the 230-bp polylinker. This allows for the linking together of up to eighteen different gene cassettes prior to transformation into the target organism.
Plasmid vectors form one of the central cores of molecular biology by allowing the manipulation of natural and synthetic DNA molecules. The exploitation of current vectors, which are of limited capabilities, has led to the need for new and inventive vectors which increase the maneuverability of DNA. The pMeca plasmid is a computer designed synthetic DNA vector system that allows for the ability to transform an organism with multiple genes making it easier to stack “transgenes” together or engineer novel metabolic pathways. The plasmid is ideal for large scale applications, speeding up the generation of transgenes and simplifying discovery and development work, with a minimal number of steps.
From the laboratory of Wayne A. Parrott, PhD, University of Georgia.
|Accession ID:||AF017063 (for pMeca multiple cloning site)|
|Tested Applications:||Development of novel transgenic crops and transgenic bacteria for multiple uses in the biotech and pharmaceutical industries|
|Grow in E. coli at 37 C:||Yes|
|Cloning Site 5':||EcoRI|
|Cloning Site 3':||HindIII|
|Vector Backbone and Size:||2860 bp|
|High or low copy:||High|
|Shipped:||Ambient temperature, spotted filter paper|
It is suggested to first plate the transformed cells on LB/Amp with IPTG and X-gal. Once familar with the growth characteristics, then remove the use of IPTG and X-gal. Bacteria transformed with the vector will also take longer to appear blue. One trick is to place the plates in the refrigerator (after growth at 37C) for several hours to better see the blue phenotype. Also, it is easy to pick based on size, so it is not necessary to wait a long time for the small colonies to grow or become blue; just select the large colonies on the plate after 16 hrs of growth. While it is not necessary to use chloramphenicol for the production of plasmid, some may find it necessary.
Strain DH10B has been reported to not be transformable with pMECA. That appears to not be the case once pMECA has an insert. This can be an advantage in that only pMECA clones containing an insert grow well in this strain. It is recommended that the vector first be transformed into DH5alpha to grow up stock plasmid. Then this stock can be used to clone into and transformed into DH10B. In theory only the clones that appear on the plate should be recombinant.
(A) pMECA colonies with (large and white) and without (small and blue) inserts. (B) pMeca Plasmid Map. The multiple cloning site is revealed as an EcoRI/HindIII fusion with the lacZ gene. Unique restriction sites are shown, and the same numbers are used for neoschizomers (isoschizomers that cut at different sites within the same sequence).
pMeca Plasmid characterization
pMeca Plasmid utilization
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