bioreactor studies of heterologous protein production by ...

stiU deleted hm the plasmid. Such plasmid-bearhg tells can pw in YNB selective
medium but are not able to produce glucoamylase. Therefore, afar severai Wers on
the agar slants containhg YNB selective medium, recombinant ceUs will gradudy lose
their ability to produce giucoamylase. In order to overcome this problem, maltose
selective solid agar medium (medium V) was used to select and maintain this recombinant
strain. Since the host celi strain C468 camh a mutation blocking the utilhion of
maltose as carbon source. the presence of the plasmid which contains the glucoarnylase
gene is neccssary for the host ails to grow on maltose. The maltose seleaive medium
imposes double seleetion pressure (Iack of leucine and the use maltose as carbon source).
Only cek that contain plasmids with both LEU2 and giucoamyiase genes are able to grow
on the maltose selective medium ïndependentiy. In the screening process, ceb hm the
original strain were diluted in sterile water to obtain individual colonies and then the
diiuted celi solution was spread ont0 aga plates containhg maltose selective medium to
obtain individual colonies. Each colony fomed on the above agar plates contained both
LEU2 and glucoamyhse genes. Such screened colonies are refemd to as the selected
strain. Th.& screening process was camed out every thne months and the selected
recombinant cek were rnaintained on the maltose selective solid agar medium in order to
keep the biological characteristics invariant