An Introduction to lInCh

Lambda InCh vectors are bacteriophage lambda ( l) derivatives which facilitate transfer of cloned DNA from pBR322-type or pUC-type plasmids to the E. coli chromosome. InCh stands for "Into the Chromosome" The transfer depends on 3 regions of homology, at which recA dependent recombination is the mechanism for genetic exchange and on one site specific recombination event at the bacteriophage lambda attachment site, att.

The first of these recombination events occurrs during growth of the phage in a cell containing a plasmid. Recombination at one or the other of two regions shared by the plasmid and the phage results in cointegrate formation. Resolution of the cointegrate at the other region then results in transfer of genetic material from the plasmid to the phage. The second step can happen either during growth of the phage or at a later step. This is illustrated below. The two homologous regions are part or the bla (Ampicillin resistance) gene of pBR322 and a region near the pBR322 replicative origin we call "near-ori". This second region is present in most pBR322 derived vectors but is inert.

Fig1. Homologies

The relevant portion of the phage genome is shown at the top with the two regions of homology, "Near-ori" and "'bla IG" represented by double thickness lines. The plasmid vector pBAD18 is represented below linearized near the origin of replication. Regions of homology are indicated by the diagonal lines. Between the near ori and ‘bla regions of pBAD18 are, araC, the araBAD promoter, the multi-cloning site (indicated by the uppercase letters below the line representing the plasmid) and the part of the bla gene deleted in the phage above. By a double recombination event an active bla gene and the ara expression system can be recombined onto the phage. At the bottom the scale in basepairs is given.

Once the plasmid insert has recombined into the phage, replacing the KanR allele with a comnplete bla allele along with whatever is cloned between the two homologous regions, an Ampicillin resistant lysogen can be selected. This involves site specific recmbination at the lambda attachment site, att, on the E. coli chromosome. This is illustrated in step 2 in the figure below.

Finally, nearly all of the lambda DNA is removed by another homologous recombination event. An 800 bp fragment of the chromosome right next to the att site is cloned in the phage so that in the lysogen there is a direct repeat. Recombination between these regions loops out the intervening sequence deleting it. This is easily selected since the phage has a temperature sensitive repressor, cI857, and is induced at 42 C killing any cell in which the deletion has not occurred. This results in a strain with a stable single copy of the insert from the plasmid on the chromosome.

  

The three steps involved in transfer of an expression system from a pBR322 derived plasmid to the chromosome in stable single copy. The lInCh chromosome is shown as the outside circle at the top. The plasmid is shown as the inner circle. In the first step, recombination at both the 358bp 'bla region and the 409 bp near-ori (n.o.) region results in transfer of the promoter-gene expression system and a functional bla (AmpR) gene to the phage replacing the KanR gene. In the second step a lysogen is formed by site specific recombination at the att site conferring ampicillin resistance and temperature sensitivity. The orientation at the att site is indicated relative to the flanking markers gal and bio. In the third step, recombination in the 800 bp direct repeat region, near-att (n.a.) removes most of the phage DNA conferring temperature independence. The att region in the stabilized strain is shown at the bottom. 

Created by  Dana Boyd  on Monday, January 25, 1999. 
For more information or if you want the strains, please contact Dana Boyd.