Week 5

Next Generation Synthesis: Changing the color-generating chromophore of the purple Acropora millepora chromoprotein (amilCP)

Week 5: Next Generation Synthesis

Part A: Preparing backbone and chromophore insert

This week we changed 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. Their strategy to identify where to mutate amilCP was inferred by sequence similarities to the chromophore region that allows for spectral engineering of the structurally-characterized and well-known green fluorescent protein (GFP), which is native to the jellyfish Aequorea victoria.

1. pUC19 cut with PvuII

We digested the pUC19 plasmid to generate a backbone where we will insert our chromophore gene.



2. Extract muAV plasmid

Then we mini-prep the mUAV plasmid from overnight cultures obtaining 2 samples of 28.7 and 25.4 ng/ul


3. Design primers for muAV PCR

In the mUAV plasmid, we wanted to identify the regions of the gene that we needed: promoter, RBS, coding sequence, terminators. The promoter was not annotated in Benchling, so I did a blast to find it

Now we needed to design primers to generate amplicons of the gene that we want to insert in the pUC19 backbone. This primer design will be key because we will be able to introduce substitutions that will change the chromophore color.


For the first amplicos, we want to generate a FW primer that contains the region in pUC19 right before the PvuII cut in bp 54 and the first nucleotides of the promoter in the chromophore gene in mUAV The reverse primer will be formed by bps before the chromophore region

For the second amplicos, we want to generate a FW primer that contains the 24bp before the chromophore region, the mutated chromophore region and some extra bp after that The reverse primer will be formed by last bps of the terminator and beyond appended to 17bp right afther the PvuII cut in pUC19(after 377)

4. Generate amplicons through PCR

Once we have our primers, we can perform a PCR to amplify the specific gene fragments that we want, and introduce substitution in the chromophore sequence

Part B

Gibson assembly

5. Ligate pUC19 backbone with our inserts to obtain a plasmid with a modified chromophore gene

Part C

Electroporation and results

6. Transform cells through electroporation

We peformed the electroporation of the plasmid with our gene insert. The time constant that we got however was around 3.6ms instead of the expected 5ms. I obtained some purple colonies, but not the rainbow of colors that I was expecting. I believe the problem happened during Gibson. Since I did not have enough insert, the efficiency of the reaction might have been lower and some of the plasmids did not get the insert gene.

Part D

Discussion questions

What are the Gibson overlap sequences in our DNA assembly design? How is an overlap created between the cut pUC19 and the PCR amplified parts of the amilCP gene? What is the melting temperature for each overlap sequence and how do they compare to the incubation temperature for the Gibson assembly reaction? See http://biotools.nubic.northwestern.edu/OligoCalc.html What is the purpose of each enzyme in the Gibson assembly mix? Our 50 ul PCR reactions included 0.5 uM of both forward and reverse primer and 200 ng of template DNA in the form of a 2,924 bp plasmid. Assuming perfect doubling of DNA copies with each PCR cycle, how many cycles does it take to use all primers? Recall, primers are extended to form copied products. See https://nebiocalculator.neb.com After the electroporation step in our experiment, how can we select for bacteria that contain only the original mUAV plasmid? Read the following post and its references: https://bitesizebio.com/2267/plasmid-retention/ Comment on the likelihood of a colony containing two plasmids from our electroporation into NEB 10-beta cells.