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I. INTRO

We will be changing 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.

First we will prepare for a Gibson assembly by using polymerase chain reaction (PCR) to produce two sets of amplicons as inserts and a restriction digest of the common cloning plasmid pUC19 to produce a new backbone (i.e. origin of replication and drug resistance gene). As template, both reactions use the amilCP-encoding plasmid that was miniprepped from the Addgene mUAV sample (deposited by the Nakayama lab at the University of Edinburgh and related to their paper on Mobius Assembly via a Mobius Assembly Universal Acceptor Vector). One set of amplicons copy the region of the amilCP gene that precedes the chromophore, including the transcription promoter and translation ribosome-binding site (RBS). Another set amplicons copy the region that spans 24 basepairs before the chromophore to just beyond the gene's transcription terminators. The latter includes a diversified chromophore-coding segment dictated by mismatches in the PCR primers with respect to the mUAV DNA template. The amplicon sets both include one end that overlaps by 20-22 bases with distinct ends of the large backbone fragment from the pUC19 digest. Lastly, we will express our colorful variety of amilCP mutants in chemically competent E coli cell.

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II. WORKFLOW

1. Setup 5.1 ml overnight cultures with conditions defined in the table below. For each condition, combine 5 ml of the specified media (already supplemented with chloramphenicol) and 100 ul of E coli from the starter culture with the specified plasmid content. Prepare duplicates for each condition for 24 new subcultures samples in total.

Plasmid Content                     Growth Media                     Growth Temperature

‍pAC-LYCipi                                  LB                                                 30C

pAC-LYCipi                                  LB+Fructose(6g/L)             30C

pAC-LYCipi                                  2YT                                              30C

pAC-LYCipi                                  2YT+Fructose(6g/L)          30C

pAC-LYCipi                                  LB                                                  37C

pAC-LYCipi                                  LB+Fructose(6g/L)             37C

pAC-LYCipi                                  2YT                                              37C

pAC-LYCipi                                  2YT+Fructose(6g/L)          37C

pAC-LYC                                       LB                                                  37C

pAC-LYC                                       LB+Fructose(6g/L)              37C

pAC-BETAipi                              LB                                                  37C

pAC-BETAipi                              LB+Fructose(6g/L)              37C

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2. Grow cultures for 20-24 hours in the circular roller drum (set to 7) within the appropriate warm room for each condition.

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3. Transfer 200 ul of each grown culture into an empty well on a clear-bottom 96-well plate. Transfer another 200ul from each grown culture into a different empty well on the same plate.

‍These should fully occupy rows A-D on the plate and will be used for estimating cell growth by optical density at the wavelength 600 nm (OD600). At 600 nm, dense cell suspensions will scatter light and the background absorbance of the media is minimal. See OD600 to cell count conversion.

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4. For each sample, concentrate a pellet. Make sure the bacteria within the sample are at homogenous density; Fasten the culture tube's cap tight and vortex sample as necessary.

4.1 Using two 700 ul transfers, transfer 1400 ul of each grown culture into a new 1.5 ml Eppendorf microcentrifuge tube.

4.2 Centrifuge at 13-14,000 rpm for 1 minute.

4.3 Discard supernatant. Use the pipette to remove remaining supernatant.

4.4 Repeat the {transfer, spin, and discard} twice more per sample to add onto its pellet.

4.5 Use the pipette to remove remaining supernatant

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5. Review acetone safety data sheet. Note, acetone is compatible with polypropylene, which is the plastic makeup of the 50 ml conical tubes and 1.5 ml Eppendorf microcentrifuge tubes.

Apply 300 uL acetone to each sample's pellet and pipette up and down until the pellet is resuspended. The acetone will precipitate cellular proteins and the carotenoid pigment will go into solution. See organic solvent precipitation slides.

6. Isolate acetone-dissolved pigment from solid cellular precipitate.

1. Centrifuge the acetone resuspended pellets at 13-14,000 rpm for 1 minute.
2. Save the pigmented liquid portion by transferring 250 ul of supernatant into a fresh 1.5 ml Eppendorf microcentrifuge tube. Carefully avoid disturbing the pelleted precipitate.
3. Dilute the transferred supernatant with 250 ul of water. This is to avoid acetone corrosion of the polystyrene plates in the remaining steps.
4. Transfer 200 ul of diluted pigment extraction into an empty well on the same clear-bottom 96-well plate as before. Transfer another 200 ul of diluted pigment extraction into a different empty well on this same plate. These should fully occupy rows E-H on the plate and will be used for estimating lycopene and beta-carotene yield based on their respective peak absorption wavelengths of 474 nm and 456 nm. See lycopene spectrum.

7. Measure the absorption spectrum of all wells on the full 96-well plate using the BioTek plate reader. Note, absorbance is measured as 2 - log10 %transmittance. See the Beer-Lambert Law.

1. Measuring absorbance in the 350-650 nm UV-Vis spectrum at 2 nm increments.

8. Final color plate:

8.1 Whatman paper tests

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