and l211a similar to those in the previously described dmlt norton eb lawson lb freytag lc clements jd clin vaccine immunol 2011 april 18 4 546 51 seq id no 5 in m51559 in addition to the two mutations in m51405 there are mutations in the region 189 to 197 in dmcta the sequence is nap g ssmsn seq id no 8 whereas in mmcta the sequence is dss g titgd seq id no 9 the underlined residue in each sequence is the r192g mutation present in both molecules the reinsertion of the mutant ctxa genes was done by gene replacement using the above described suicide plasmid pmt suicide sacb a native ctxa gene together with a fragment of the upstream zot gene was pcr amplified from chromosomal dna isolated as described previously from strain phil6973 an o1 el for clinical isolate of v cholerae using the primers zot f 5 u2032 gggggtctctctagaatgctgcgggagcaaggcggctg 3 u2032 seq id no 10 and cta r 5 u2032 ggggggaagcttataattcatccttaattctattatgtgtatcaatatcagattg 3 u2032 seq id no 11 see table 1 the resulting fragment was digested with eco31i and hindiii and inserted into a p15a based expression vector digested with xbai and hindiii in order to generate the double mutant cta with amino acid changes r192g and l211a a dna fragment was synthesized the allowed the entire 3 u2032 end of.