Part:BBa_K1131000:Experience

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Pathway for BBa_K1131000

As mentioned, this FMO part can be used to produce indigo from indole. Specifically, this is the pathway being employed:

Upon cloning this enzyme into E. coli with the BBa_J23100 Anderson Promoter, and adding tryptophan (a precursor to indole) in the LB media, we observed a dense blue color in our cultures.

We proceeded to characterize this enzyme in a variety of ways.

Characterization

Detection

We wanted to confirm that we were producing appreciable amounts of indigo compared to other side products. When indoxyl dimerizes to produce indigo, a few other side products can be generated as well such as

We ran standards for indigo, indirubin, and isatin, and some of our bio-indigo producing E. Coli lysate on the HPLC and LC-MS. The observed peaks for each of these products are shown below.

We can tell that we are producing appreciable amounts of indigo compared to other side products in the cell.

Titer

Our next step was to measure the amount of indigo we are producing. We used the same plasmid as above to try and constitutively express large amounts of FMO. We tried a variety of conditions to try to maximize our indigo production as shown below:

LB stands for standard LB Broth as growth media, while YE refers to Yeast Extract. With increasing concentrations of added tryptophan (Trp), we are able achieve corresponding increases in indigo production as measure in mg/L on the Y-axis. Refreshed tryptophan media indicates that after 12 hours of growth, bacteria were supplied with fresh growth media containing the same amounts of tryptophan added as before. Though other conditions such as varying salts (NaCl vs KCl) thought to affect tryptophan transporters and buffering conditions were tested to see if they affect indigo production, the largest effector was clearly the amount of tryptophan. This makes sense as tryptophan is the raw material used by E. coli to produce indole, the naturally occurring substrate used by our heterologously expressed FMO.

Toxicity

First, we wanted to answer if the generated indigo is toxic to our bacteria. If so, large scale production of indigo may be difficult. To do this, we did a time course experiment in which we compared the number of bacteria on a hemocytometer. The two constructs we compared were:

The mFMO part has been mutated in three places to eliminate all indigo-producing activity. The construct containing mFMO was used as a negative control as it is almost the same construct except for the three mutations specified on the mFMO part page. Indigo production in bacteria containing either of the two plasmids was measured over the course of 24 hours straight at 30C in plain LB media with .74g/L added tryptophan. The results are shown below.

From this, there is no clear indication that indigo is toxic to the cell.

Kinetics

Lastly, we wanted to measure how quickly FMO was able to generate indigo from indole. We used Michaelis-Menten kinetics to model the behavior of FMO with various concentrations of indole substrate to determine the Km and Vmax of the enzyme. To do this, we first needed to obtain purified FMO enzyme. We used the T7 expression system and chemically competent BL21 cells to express a His6 tagged FMO and purify it using a nickel column. The plasmid we used is depicted below.

We then generated a Lineweaver-Burke plot and a time vs. rate plot to describe FMO kinetics. Note that our data informs about the rate of indigo generation and not indoxyl, and thus we are not specifically calculating the rate of oxidation from indoxyl to indigo. File:FMOKineticsB.jpg

From this we established a Km of 1.30mM for FMO and Vmax of about 1.59e-5 mM/s.

Other Information

BBa_1131001 - mFMO

We refer to part BBa_1131001 in our toxicity experiment as the mutated, knocked out version of FMO. This part can be found here.

Citations

Pinero-Fernandez, S. "Indole Transport Across Escherichia Coli Membranes." Journal of Bacteriology. 193.8 (2011): 1793-1798. Print.

Kim, Si Wouk. "Bio-indigo production in two different fermentation systems using recombinant Escherichia Coli cells harboring a flavin-containing monooxygenase gene (fmo)." Process Biochemistry. 46. (2011): 788-791. Print.

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