Results
Plasmids were ordered from Addgene.org and grown in LB broth. Both tsPurple and asPink are constituatively expressed. Cells were collected and the plasmids were isolated using the BioBasic DNA isolation kit.
The DNA was run on a 1% agarose gel and then stored at -20C for future use.
Several attempts were made using the mutagenic primers and the asPink plasmid to produce new mutated plasmid with the appropriate changes.
Using the Q5 DNA polymerase, we were successful in completing the SDM reaction to produce the new pSB1C3-asPinkTT9495QI.
Transformation into DH5alpha resulted in several pink colonies on selective media. Individual colonies were selected for DNA isolation, Hyp188III digestion, and sequencing. It was found that all colonies selected had the correct digestion pattern. When the DNA was sent for sequencing the correct DNA changes were found.
We were able to change T94 and T95 in asPink to the corresponding amino acids Q94 and I95 of tsPurple. The change did not result in a colour shift, as the mutant protein was still pink, but it did result in a much slower maturation of the protein. Colour was only observed in the bacterial colonies after more than 24 hours and a period of incubation at 4 degrees C.
The next steps in the lab will mean designing additional mutagenis primers to change any other identified amino acids to those in tsPurple. This will be a long process.
The asPink gene should also be placed in a plasmid that has a purification tag. This would allow us to purify and test the protein in the future.
Engineering of P. denitrificans should begin. First, to test if the chromoprotein can be expressed in the bacteria and the colour observed. Second, to test how the engineered strain competes against the other naturally occuring bacteria in the filtration system. Finally, to see if this bacteria is able to detect any pathogens in the aquatic system. More info on biosafety measured can be found here.
