We discovered an MFS transporter in the same operon as ismA, the enzyme catalyzing the first step of the cholesterol to coprostanol pathway. Believing it is involved in cholesterol transport, we decided to clone it into B. subtilis using promoters and integrative vectors from LMU-Munich 2012's B. subtilis BioBrick Box.

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Since flow cytometry with BODIPY cholesterol didn't work, we decided to run a cholesterol uptake assay instead. We measured the cholesterol content of the growth media (supplemented with cholesterol) as the bacteria grows. Decrease in cholesterol content in the media indicates cholesterol uptake by the bacteria.

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We designed a new cell culture medium that contained 0.1% w/v lecithin. Lecithin is commonly used in industry to emulsify fats, and it would emulsify the cholesterol, allowing it to stay in solution and be accessible to the bacteria as they grow.⁶This media would be used for another cholesterol uptake assay. Additionally, we decided not to pellet out the bacteria as the quantification kit would not be able to access the cholesterol inside of the bacteria, still quantifying only the cholesterol in solution.

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We decided to greatly lower the amount of cholesterol added to each bacteria lecithin culture from 2 mg/mL to 80 μg/mL. This would mitigate the issue of solubility and be 10 times more concentrated than the standards for a fluorometric assay, which is much more sensitive. Additionally, bacteria would not interfere with a fluorometric assay, which is possible with the cholesterol quantification kit, so we opted to try that instead.

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We wanted to confirm that our probiotic bacterium could grow in conditions similar to the gut. The first step was to test if B. subtilis could grow in an anaerobic environment, since the gut lumen itself lacks oxygen.

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The first step of our project was to clone the genes of our enzymatic pathway into E. coli and determine the best conditions for expression. We decided to clone each gene into a separate bacteria and attach His-tags so that the proteins could be isolated.⁸ We put the genes under the control of the inducible lac operator so that we could test the different IPTG induction conditions.⁹

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We hypothesized that ismA was trapped in the inclusion bodies, so we cultured ismA for 4 hr at 37˚C, 16 hr at 28˚C, and 24 hr at 16˚C. Samples from every stage of the extraction and purification process were tested on a western blot.

ismA was expressed in the cell lysate in all three conditions, but absent from the supernatant. This confirmed our suspicion that the ismA was stuck inside the inclusion bodies.

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ismA was denatured with 6M guanidine-HCl instead of 8M urea.

We also decided to perform a buffer exchange from guanidine-HCl to 80mM of Tris-HCl gradually in 30 minute increments to minimize incorrect folding during renaturation.¹¹

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To minimize the possibility of ismA being aggregated despite having the correct sequence, we decided to add a SUMO tag to ismA, which would help solubilize the protein.¹³

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We used Gas-Chromatography Mass-Spectrometry (GC-MS) to test our purified proteins because of its sensitivity and molecule identification size range. First, we needed to create standards for our 4 sterols. This would allow us to compare our reaction chromatograms to our standard chromatograms to identify which peaks correspond to which molecules.

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To isolate our proteins to the highest degree possible, we designed an in vitro assay protocol. The paper “Substrate specificity and inhibitor analyses of human steroid 5ß -reductase (AKR1D1)” by Chen, Drury, and Penning performed a very similar assay, which we used as a template for our own set of substrates and enzymes.¹⁴ Our strategy was to build a reaction pool that would contain everything except the sensitive cofactors and the proteins. These would be added directly before incubation.

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The organic solvent is present in the mixture to dissolve the hydrophobic sterols. We designed a strategy to test various other water-miscible organic solvents at varying concentrations. We needed an organic solvent which would dissolve all 4 sterols, and at final concentrations low enough so not to harm the proteins' tertiary structures during the reaction.

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To assess the efficiency of the enzymes in vitro, 4 preliminary assays were designed: 2 assays for each enzyme AKR1D1 and AKR1C4 (at this point, ismA had still not been properly purified). Further, to investigate the effect of competitive substrate inhibition, two different substrate concentrations were tested: 50µM and 100µM.

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Once ismA had been purified using SUMO protein fusion, it was possible to run in vitro assays using ismA and to test our entire reaction pathway. In theory, the ismA step of the pathway has already been verified, which serves as a way of ensuring that our assays are valid.

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To improve the catalytic activity of AKR1D1, we needed to test a series of promising mutant AKR1D1 proteins compiled by the dry lab team. However, protein purification is a long and resource-intensive process. We designed a new type of protein assay titled “pop assay”.

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While it was encouraging to confirm the functionality of each step on the GC-MS, AKR1D1 still did not display the activity required by our project. Aside from the mutant assays we had planned, we brainstormed elements of our in vitro assay which could be improved or optimized to yield more product. The first factor we experimented with was derivatization.

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We tried adjusting a few other variables in our in vitro assay protocol. Eventually we knew that we would have to run enzyme kinetics experiments, so we decided to run some preliminary tests at lower incubation times to see if the product was still detectable. Additionally, we attempted doubling the enzyme, cofactor, and substrate concentrations to see what would happen.

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Using our pop assay protocol, we designed a new line of experimentation revolving around the mass testing of promising AKR1D1 mutant proteins. The first batch of mutants tested via the pop assay contained a total of 13 mutants.

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After testing cholesterol uptake in B. subtilis and enzyme function, we needed to clone our pathway into B. subtilis. We had three integrative vectors at our disposal: pBS1C (amyE integration), pBS4S (thrC integration), and pBS2E (lacA integration).¹ We designed primers for Gibson assembly to insert all three genes of our pathway; ismA, AKR1D1, and AKR1C4; into pBS1C under the control of the very strong constitutive promoter Pveg.1 Overhangs were included in the primers to insert ribosome binding sites ahead of each gene, and a terminator at the end. Ribosome binding sites were predicted using the “Design Operon” function on iDOG.²¹

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We previously saw through a western blot that not all proteins of our pathway were getting expressed when they were combined into a single operon. We designed primers to use Gibson assembly to insert each gene into their own integrative vector under the control of the very strong constitutive promoter Pveg. ismA would go into pBS1C (amyE integration), AKR1D1 into pBS4S (thrC integration), and AKR1C4 pBS2E (lacA integration).³ Like before, overhangs were included to introduce ribosome binding sites and a terminator. Ribosome binding sites were predicted using the “Design Operon” function on iDOG.

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To test our proof of concept, we decided to use the pop assay protocol which we have developed. If it is functional, we would expect to detect the final product as well as the intermediates on the GC-MS.

We popped B.subtilis bacteria with our entire enzymatic pathway integrated under one operon, which should theoretically be able to convert cholesterol into coprostanol.

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Because having the entire pathway integrated in one operon and under one promoter did not work, we opted instead to integrate only one protein under a single promoter, which could potentially resolve the expression problems. To test this, we first needed to first run pop assays on B. subtilis containing ismA, as well as one containing AKR1D1 only. If we verify that this works, we can run a pop assay on B. subtilis containing all three enzymes down the road.

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