1) Generally, PKS clusters use conserved functional domains and you can even deduce cluster organization from the antibiotic structure. it's highly possible that you will get some positive clones from the library. Actually, you can use some PKS fragments from other strains as probes directly. One thing should be considered is that one strain may harboring multiple PKS clusters, which will of course complicate your project.
2) The positive result of heterogeneous expression would be most reliable. However, it may be difficult since you cluster is as huge as 56kb. To do this, Bac based library seems necessary and a potent genetic manipulation system (tranformation, transfection or conjugation) would be essential to deliver whole cluster into host. Also, the host should also possess all transcriptional factors needed for this cluster. So, i think gene disruption or displacement experiment may be preferred to identify the edges of the cluster, if your strain is not very refractory to foreign DNA and your antibiotic is easy to be characterized.作者: any333 时间: 2014-2-3 18:38