3. Problem
• Which of the 8 secretory proteins of B. subtilis
NZ8900 is/are found in large amounts in the
growth medium?
4. Methodology
1. 8 genes cloned using SURE system
overexpression vector pNZ8902 or pNZ8901
2. Genes were fused to C-terminal 6His-tag
encoding sequences for imunnodetection
3. Bacterial cultures grown to midexponential
phase and gene expression was induced with
subtilin
4. Testing protein production (whole cell extract;
extracellular; membrane; cytoplasmic) and
Microarray analyses
7. Conclusion
• Data reveal possible bottlenecks in the
protein overproduction process
• Recombinations in the genes controlling the
expression of XynA and NprE would produce
valuable proteins in high amounts fit for
industrial production
8. Microarray Analyses
• measure the expression levels of large
numbers of genes simultaneously or
to genotype multiple regions of a genome
9. General Effects of Overproduction
• Downregulation of the sdpABC (sporulating delay
protein) operon
– Role in apoptosis
– Kills non-sporulating siblings and feeding on the dead
cells under conditions of nutrient limitation
• Upregulation of class I heat-shock genes coding
for molecular chaperon groES and groEL
– Protects the cell from toxic accumulation of mis- or
unfolded protein
