This method provides an efficient alternative to the conventional analog-based approach in obtaining amino acid overproducers. This technique outcompetes the traditional analog-based method by providing accuracy, sensitivity, and high throughput simultaneously. This method sheds new light on understanding amino acid overproduction strengths and the translation of rare codons and could, theoretically, be applied to all microorganisms.
This protocol relies mostly on basic molecular experimental operations which are easy to follow but may require multiple trials in order to achieve the suitable selection of straining stringencies. A visual demonstration of this technique offers a direct appreciation on the performance of the selection and a screening system in identifying amino acid producers. To begin this procedure, add the vector in the marker fragments, on ice, in a molar ratio of one to one to seven point five microliters of assembly mix to a total volume of ten microliters.
Incubate at 50 degrees Celsius for one hour. Transform five microliters of the assembly product into 50 microliters of component cells at 42 degrees Celsius for 30 seconds. Recover the cells in Super Optimal broth with Catabolite repression medium at 37 degrees Celsius for one hour.
Then, plate them on LB Agar medium and incubate overnight at 37 degrees Celsius. The next day, use a preferred commercial kit to isolate the plasmid. First, transform 50 microliters of the competent cells with one microliter of plasmid that carries the wild-type KanR.
Transform a second set of the competent cells with the plasmid containing KanR RC 29 with all leucine codons replaced by the rare codon CTA. Plate and incubate the cell culture as outlined in the text protocol. Next, transfer the colonies that harbor the wild-type selection marker gene in the rare-codon-rich derivative individually into five milliliters of five-fold diluted LB medium containing kanamycin at a concentration of 50 micrograms per milliliter.
Incubate the samples in a shaker at 37 degrees Celsius while shaking at 250 rpm. Then, transfer 200 microliters of each of the cell cultures into a 96 well plate in triplicate at defined time points. Use a plate reader to measure the OD600.
Inoculate the stains harboring the KanR RC 29 marker gene in two sets of five milliliters of the 0.2 x LB medium that contains kanamycin, with or without a supply of L-leucine. Add L-leucine to one of the media to a final concentration of one gram per liter. Incubate the samples in a shaker at 37 degrees Celsius with shaking at 250 rpm and measure the OD600 for each culture at defined time points.
To begin, transform 50 microliters of the parent strain used for mutagenesis with one microliter of plasmid that carries the wild-type GFP or the wild-type PPG. Plate and incubate the cell cultures as outlined in the text protocol. Next, transfer the colonies individually to five milliliters of the properly diluted LB medium.
Incubate the samples in a shaker at 37 degrees Celsius with shaking at 250 rpm. For fluorescence markers, transfer 200 microliters of the cell cultures into a 96 well clear bottom back plate in triplicate at defined time points. Measure the OD600 in the fluorescence and calculate the fluorescence intensity.
For chromogenic markers, measure the color development of the cell cultures. Perform the feeding assay as previously described and measure the fluorescence intensity or the color development at defined time points. First, transform 50 microliters of the mutant cells with one microliter of the plasmid carrying the selection marker KanR RC 29, or the screening markers GFP RC or PPG RC.Next, centrifuge the cell cultures at 4000 times G for five minutes.
For selection, discard the supernatant and add five milliliters of 0.2 x LB medium containing kanamycin to cells carrying the selection marker. Incubate the sample overnight in a shaker at 37 degrees Celsius. The next day, plate the overnight culture onto 0.2 x LB agar medium containing kanamycin and incubate at 37 degrees Celsius for 12 hours.
For screening, plate the appropriate number of cells harboring the screening marker onto LB agar medium containing the appropriate antibiotic. Incubate at 37 degrees Celsius for eight hours. After this, inoculate the LB medium containing the appropriate antibiotic with each single colony from the plate.
Incubate the samples in a shaker at 37 degrees Celsius while shaking at 250 rpm. Measure the OD600 and the fluorescence and calculate the fluorescence intensity if GFP RC is used. Measure the color development of the cell cultures if PPG RC is used.
To verify the amino acid productivities of the candidate strains, prepare seed culture by inoculating five milliliters of LB medium with each of the candidate strains and let the cells grow overnight in a shaker at 250 rpm and at 37 degrees Celsius. The next day, harvest the cells from one milliliter of the cell culture by centrifuging at 4000 times G for two minutes. Discard the supernatant and resuspend the palette with one milliliter of sterile water.
Inoculate 20 milliliters of M9 medium containing four percent glucose with 200 microliters of the cell suspension and incubate in a 250 milliliter shaker at 250 rpm and at 37 degrees Celsius for 24 hours. The next day, centrifuge one milliliter of the culture medium at 4000 times G for five minutes. Transfer 200 microliters of the supernatant into a clean 1.5 milliliter tube.
Prepare L-leucine standard solutions at the concentrations shown here. In a fume hood, add 100 microliters of one millimolar triethylamine and 100 microliters of one molar phenyl isothiocyanate to both the supernatant and the standards. Mix the solutions gently and incubate them at room temperature for one hour.
Then, add 400 microliters of n-hexane to the same tube and vortex it for ten seconds. The lower phase contains the amino acid derivatives and is used for HPLC analysis. Filter the lower phase through 0.2 microliter polytetrafluoroethylene membranes.
After this, run one microliter of the sample on an ultra HPLC equipped with a C18 column with a flow rate of 0.42 milliliters per minute and at column temperature for 40 degrees Celsius. Use a diode array detector to detect the targeted amino acids at 254 nanometers and calculate their concentrations by mapping the peak areas under the standard curve. In this study, amino acid overproducers are identified by using rare codon-rich markers to simultaneously achieve accuracy, sensitivity, and high-throughput.
For the selection system, a sharp decrease in OD600 for strains harboring the rare codon-rich antibiotic resistance gene should be observed in comparison to the strain harboring the wild-type. The inhibition of rare codon on protein expressions mostly takes place under starved conditions. After extra feeding of the corresponding amino acid, the OD600 for the strain harboring the rare codon-rich antibiotic resistance gene increases significantly.
For the screening system, the fluorescence intensity in the number of fluorescent cells are significantly lower for the strain that expresses the fluorescent protein from the rare codon-rich gene than from the wild-type gene. Feeding of the corresponding amino acid will restore protein expressions from the rare codon-rich genes. When using the purple protein, the color developed from the rare codon-rich PPG is lighter than that from the wild-type gene.
The undiluted LB medium allows slow expression of the rare codon-rich PPG without L-leucine feeding in the expressed purple protein becomes visible once the cells are pelleted. However, this does not conceal the fact that gene expression from the rare codon-rich PPG is enhanced by feeding of the L-leucine. The rare codon-based strategy could identify overproducers of the targeted amino acids from the mutation library and these mutants should produce higher amounts of the targeted amino acids than the parent strains.
When attempting this procedure, it is important to adjust rare codon frequency to achieve a clear discrimination in the OD or color between cells harboring the wild-type markers and the rare codon-rich derivatives. Transcriptome analysis and the genome sequencing could be applied to the selected strains to investigate the mechanism related to amino acid productions. Using this technique, amino acid overproducers could be efficiently identified and analysis of their genetic information would offer new insights into their mechanisms behind amino acid overproductions.
Amino acid derivatization could be harmful. Make sure to wear gloves and the protective clothing and perform these steps in a fume hood.
