Next Generation Synthesis
In this week, we tried to change the color-generating chromophore of the purple Acropora millepora chromoprotein (amilCP) to a variety of orange, pink, and blue mutants. These divergently-colored genetic variants of amilCP were described by Liljeruhm et al in 2018.
1. Digest the pUC19 plasmid with the restriction enzyme PvuII to generate the linear blunt-ended backbone fragment.
Green: pUC19 plasmid
Right: Buffer CutSmart
White: DNase/RNase-Free Water
NEB PvuII-HF(restriction enzyme)
Incubate reactions at 37C for 15 minutes in thermocycler
2. Prepare PCR fragments for the gene assembly of amilCP mutants.
DNase/RNase-Free Water
miniprepped amilCP (blue cap)
PCR tubeI: Forward primer & reverse primer (yellow caps)
PCR tubeII: Forward primer & reverse primer (purple caps)
2X Phusion HF PCR master mix
Amplify PCR fragments in Thermal Cycler
Purify DNA fragments from previous PCR steps
Measure the concentration of purified DNA.
Then setup Gibson Assembly reaction in PCR tubes.
Heat Shock the cells to let DNA from previous Gibson Aseembly going through the membrances.
Incubate at 37C in warm room
Left: Mutagenetic cells of my plate after incubated for several days in the warm room
Right: more colorful mutagenetic cell images