recently i learn some knowledge about co-expression.
the EMBL website said, in the case of co-expression from different vectors, To ensure plasmid stability, the vectors should have:
(1)different selectable markers, usually antibiotic resistence markers.
(2)different origins of replication.
But I check some publications, they did co-expreesion from 2 vectors with different resistance markers but same replication origins. like pET16b-geneA and pET28a-geneB. or pET22b-geneC and pET28a-geneD. and both were succeed to express two protein from single colony.
so whether it means the ''different origins of replication'' is not necessary?
and in my experiment, protein PCID2 are inclusion body in several different plamid, I try to obtain soluble protein PCID2, and then i did co-expression with its binding protein Y, i co-transformed pET16b-PCID2 and pET28a-Y to E.coli, the replication origin are both pBR322, pick up single colony, Y is soluble, protein PCID2 is still inclusion body. I do not know if my co-expression succeeded or not.
Both PCID2 and protein Y were produced, so I would assume that the co-expression was successful in that regard.
If the colony was grown on a plate with both ampicillin and kanamycin then we can assume that both plasmids were present in the clone.
If you are worried that the cells have lost one of the two plasmids during shake flask cultivation, you could plate the cells after the cultivation on ampicillin plates, kanamycin plates and amp+kan plates and count the colonies. Then you will see the proportion of cells carrying only one or two plasmids.
Dear Zixun,
It is true that different origins of replication are necessary for the stability of two plasmids in a cell. Please note - two plasmids with the same ori can be co transformed. I have done that myself, and so have you. But when you propogate the co-transformants, stability of either of the plasmids becomes an issue,i.e., one of the two plasmids is removed from the cell.
In addition to Konrad's suggestion, you could also conduct colony PCR to check for both your genes of interest are in place.
I hope you are using an expression strain such as Rosetta for heterologous expression. And secondly, try using a more subtle promoter which although will lower your expression levels but may also give you PCID2 in the soluble fraction.
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