Detection of lycopene

Firstly, we should test our crtEBI enzymes, which would consequently produce lycopene, a chemical with remarkable red color. We cultured our engineered bacteria in a 96-well plate, as Figure 1 indicates, redness could be observed.

Figure 1. 96-well plate of bacteria with crtEBI. After overnight culture, the plate was spin at 4000 rpm for 30 minutes. The photo was taken after fliping the plate. Redness could be observed in most of the wells, where bactera was pellet to the bottom. Clearly, these bacteria produce lycopene.

Detection of β-carotene

After the detection of lycopene, to ensure our crtYEBI enzymes worked well, which meant a detectable amount of β-carotene should be produced, we operated High-Performance Liquid Chromatography (HPLC), for the color of β-carotene resembles the color of bacteria themselves. We cultured bacteria expressing crt genes, and extract β-carotene using acetone. As Figure 2 and 3 indicate, different amounts of β-carotene were detected due to plasmid variation (3-7 and 3td7).

Figure 2 and 3. HPLC of β-carotene The upper Figure 2 used three plasmids: CAG-MS2, BCMO, ybbO, crtYEBI; the lower Figure 3 used these plasmids: CAG-MS2, BCMO, ybbO-tdMCP, crtYEBI.

Detection of retinol

To make sure our BCMO and ybbO genes were expressed, we needed to detect retinol, which can prove the feasibility of BCMO and ybbO at the same time. We used our fully-engineered bacteria, with or without TEARS, inject HPLC with acetone extraced samples. As Figure 4 and 5 indicate, distinct peaks appeared and retinol was detected. We can witness different absorption peaks during 2 mins and 4 mins, which we reckoned represented different enantiomers of retinol. Plus, we found it hard to detect retinal, which was the product of BCMO and the substrate for the enzyme retinal dehydrogenase expressed by ybbO. We attributed the reason to TEARS, the liquid-liquid phase separator, for its strength in enriching molecules. Most retinal was gathered by TEARS, thus inverted into retinol.

Figure 4 and 5. HPLC of retinol. The upper sample were extracted from bacteria expressing CAG-MS2, BCMO, ybbO, crtYEBI; the lower from: CAG-MS2, BCMO, ybbO-tdMCP, crtYEBI.

Proof of our membrane protein induced by IPTG

β-carotene 15,15’-MonoOxygenase (BCMO) is one of the essential enzymes in our single-cell factory. We’re obliged to affirm the expression of BCMO after IPTG induction, so we ran the SDS-PAGE to confirm. As Figure 6 indicates, our bacteria didn’t successfully express a visible amount.

Figure 6. SDS-PAGE of BCMO protein Lane 5, 6, 7 and 8 show no BCMO protein has been successfully expressed whether or not induced by IPTG.

After re-checking our protocols and thoughtful reflection, we attributed our failure to the protein’s transmembrane feature, as its nine transmembrane domains might reduce the accuracy of electrophoresis. So we decided to fuse a fluorescent protein, StayGold, to our target protein and detect its expression through fluorescence imaging. As Figure 7 and 8 indicates, we got the positive result that a considerable amount of BCMO had been expressed in bacteria.

Figure 7. Fluorescent imaging confirms the expression of both BCMO and ybbO. We observed fluorescence images of E. coli (1. Bacteria expressing BCMO-linker-StayGold; 2. Bacteria expressing BCMO and ybbO-tdMCP-GFP) through an Olympus fluorescence microscope with a 1.45 NA 150× oil objective. GFP and StayGold were excited by a 488 nm laser. Images were taken with the same settings for comparison. For details about sample preparation, please check our protocol here.

Figure 8. Fluorescent imaging revealed that IPTG induction increases the expression of both BCMO and ybbO. We observed fluorescence images of E. coli (1. Bacteria expressing BCMO and ybbO-tdMCP-GFP; 2. Bacteria expressing BCMO-linker-StayGold) through an Olympus fluorescence microscope. Samples induced with IPTG have stronger signals than controls.

Gauging the efficiency of Hammerhead Ribozymes

As is indicated in many types of research, the major problem of polycistronic vectors, which contain two or more target genes under one promoter, is the much lower expression of the downstream genes compared with that of the first gene next to the promoter (Kim, Kyung-Jin et al. 2004). The tail of the coding sequence (CDS) can interfere with the head of the ribosome binding site (RBS), which can hinder RBS from combining with ribosomes. Such a shortage occurred when we assembled crtEBIY sequentially, only to find incomplete expressions of our target proteins. To optimize our parts, we adopted Hammerhead Ribozymes, which can self-cleave, thus turning a long mRNA into four short mRNAs. Each module contains a ribozyme, RBS and CDS in order. In this way, the self-interaction of the polycistron can be avoid, and every single gene can share the same translation rate. We tested the protein expressions of different combinations of two random carotene synthesis enzymes through SDS-PAGE, and as Figure 9 and 10 indicate, each enzyme can be expressed successfully.

Figure 9. SDS-PAGE of IY, EI and BE. Lane 7, 9 and 13 prove the positive expression of our target proteins induced by IPTG.

Figure 10. SDS-PAGE of IB and EY. Lane 7, 9, 13, 15 and 17 prove the positive expression of our target proteins induced by IPTG.

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