HELP!! with chloroplast transformation

[tjquinn at STUDENTS.WISC.EDU]

Hello all,

I am a rather new Chlamydomonas researcher and I need some advice from 
anyone who is willing to share their wisdom with me.

I am planning to insert a bacteria gene into the chloroplast genome. The 
bacteria gene, nifH, produces a oxygen-liable gene product, so, I am using 
non-photosynthetic mutants like FUD7 which have a major deletion at the psbA 
gene within the inverted repeat, preventing photosytem II from functioning 
(i.e. no extra oxygen).

Since the psbA gene is already missing in the FUD7 strain, it seems like a 
logical place to put the bacteria gene by using flanking sequences 5' and 3' 
from the psbA gene. 

I will be using the psbA promoter and will be using the aadA gene as a reporter.

There are a variety of ways I could construct this plasmid. I think it 
should have four elements:

1) 5' flanking sequence 
2) psbA promoter followed by nifH gene
3) aadA cassette with its own promoter
4) 3' flanking sequence


Can anyone give me some advice on the easiest way to make this plasmid?

Thanks, 

Thom Quinn

tjquinn at students.wisc.edu