The overall goal of this protocol is to describe the functional assessment of comprehensive, single-site saturation mutagenesis libraries of proteins, utilizing high-throughput sequencing. This method can help answer key questions in the fields of biochemistry and molecular evolution. Such as, what are the biochemical structural and evolutionary constraints on proteins?
And how can we engineer proteins with novel functions? The main advantage of this technique is that it maximizes a number of useful sequencing reads in a whole-protein saturation mutagenesis study, by mulitplexing library construction and sequencing. After preparing a plate of PCR Master Mix according to the text's protocol, use a multi-channel pipette to transfer 10 microliters from the 96-well plates containing the previously diluted sense mutagenesis primers to the cognate wells in the PCR plates.
Use a plate seal to cover each PCR plate, and centrifuge at 200 Gs for two minutes. Then, transfer the PCR plates to a thermocycler, and run the following program. After carrying out additional PCR and diluting the reactions, one to 100, transfer one microliter from the mixed and diluted first-round mutagenic PCR products, to the cognate wells of new PCR plates containing Master Mix.
Use a plate seal to cover each PCR plate. Centrifuge the plates at approximately 200 Gs for two minutes. Then transfer the plates to the thermocycler, and run the same program listed earlier in the video.
After verifying the PCR product sizes, and measuring the concentrations according to the text protocol, mix 100 nanograms of each NNS sublibrary PCR product into five NNS sublibrary groups. After running sublibrary PCR reactions and digesting cloning vectors in the PCR reactions, according to the text protocol, set up ligation reactions following these guidelines for each digested NNS sublibrary group, with its cognate restriction digested cloning vector. Then, incubate at room temperature for one hour.
Once electrocompetent E.coli cells have been thawed, add ten microliters of cells to each purified ligation reaction, and transfer to an electroproration cuvette. Electroprorate the cells at 1.8 kilovolts, then add one milliliter of SOB medium and incubate at 37 degrees celsius for one hour. Then, re-suspend 10 microliters of each recovery culture in 990 microliters of LB.And spread 100 microliters on LB auger plates containing 12 micrograms per milliliter of tetracycline.
Per each recovery culture, prepare a 250-milliliter culture flask with 50 milliliters of LB and 50 microliters of tet-stock. Transfer the remaining approximately one milliliter of recovery culture to a flask. Incubate at 37 degrees celsius and 200 rpm overnight.
After counting the number of successful transformants on the plates, and isolating the plasma DNA from the overnight cultures, mix together 100 nanograms of each plasmid. After transforming the DNA library according to the text protocol, dilute the library overnight culture to an OD-600 equal to 0.1. And add one milliliter to one flask.
Incubate the pre-selection culture at 37 degrees celsius and 200 RPM. Periodically monitor the growth by measuring the OD-600, until it equals 0.1. Transfer 100 milliliters of the pre-selection culture to two 50-milliliter conical tubes, and centrifuge at 4, 000 Gs for six minutes, at four degrees celsius.
Remove most of the supernatant and combine the pellets into a single, 15-milliliter conical tube. Then, after repeating the centrifugation, remove all the supernatant. To the other two flasks, add tetracycline to a final concentration of 12 micrograms per milliliter.
And a volume of the pre-selection culture, such that the final OD-600 equals 0.001. To one flask, add one milliliter of 50 milligrams per milliliter Ampicillin at a final concentration of 50 micrograms per milliliter. This is the selection culture.
While incubating the cultures at 37 degrees celsius in 200 RPM, monitor the growth of the culture containing no Ampicillin until it reaches an OD-600 equal to 0.1. Also measure the OD-600 of the selection culture. After dividing the OD-600 of the selection culture into 0.1, and multiplying by 100, transfer this volume to 50-milliliter conical tubes, and centrifuge at 4, 000 Gs and four degrees celsius for six minutes.
After removing most of the supernatant, combine the pellets into a single tube before spinning again and removing all the supernatant. To carry out high-throughput sequencing, prepare a PCR Master Mix and transfer 23 microliters to 10 PCR tubes. Add one microliter of 0.5 nanograms per microliter of purified plasmid DNA from the pre-selection culture to tubes one through five.
And from the selection culture to tubes six through 10. Mix together 50 microliters of 50 micromolar forward-orthogonal primers, OP1F to OP5F, with the respective reverse-orthogonal primers, OP1R to OP5R. In the same order, transfer one microliter to PCR tubes one through five, and six through 10.
Transfer the PCR tubes to the thermocycler, and run the following program. Then, dilute the PCR reactions 100-fold, by transferring one microliter from each tube to 99 microliters of water. Mix well, then pipette 99 microliters from the tubes, and discard.
Mix together 50 microliters of the forward, and respective reverse, primers. Then, transfer one microliter to the PCR tubes one through five, and six through 10. Then, after preparing a PCR Master Mix, add 23 microliters to each PCR tube, and run the following program.
To carry out the final PCRs to add indexing sequences, after diluting the PCR reactions 100-fold, as before, and keeping one microliter, add 23 microliters of the PCR Master Mix. For tubes with template originating from NNS sublibrary groups one through five, add 0.5 microliters of forward primers 501F to 505F, respectively. For tubes with template resulting from the pre-selection and selection cultures, add 0.5 microliters of reverse primers 701R and 702R.
Run the same PCR program as just indicated. Then, after measuring the concentrations of each reaction according to the text protocol, mix 100 nanograms of each PCR product into a single micro-centrifuge tube. After adding 6X gel loading dye, load the entire, combined PCR sample onto a two percent agarose gel.
And run the gel at 100 volts, for 50 minutes. Using a long-wavelength UV illuminator, visualize, then excise the slice containing the PCR product of approximately 360 base pairs. Purify, sequence, and analyze the sample according to the text protocol.
The plasmid map for the five modified pBR322 plasmids containing orthogonal priming sites is shown here. Testing for the specificity of the orthogonal primers using PCR, showed that the correct PCR product was only obtained when the matching plasmid was included in the reaction. And no product of any size was obtained in its absence.
Following the processing of an experiment, 6.2 times 10 to the sixth reads from the pre-selection condition, and 6.3 times 10 to the sixth reads from the ampicillin condition were obtained. The counts for each amino acid mutation from the pre-selection condition display a log-normal distribution. This figure depicts the relative fitness effect, FIA, for each mutation at each position of TEM-one.
And its distribution is shown here. Under selection at 50 micrograms per milliliter ampicillin, most mutations have a neutral, or nearly neutral, fitness effect. Corresponding to the white pixels here, and the large peak here.
A small fraction of mutations at this antibiotic concentration have substantial effects on fitness. As expected, these include mutations within the highly-conserved active site residues. In contrast, few pixels appear as significantly red.
Once mastered, this technique can be done in approximately a week. If it is performed properly, and all reagents are available. While attempting this procedure, it's important to remember to stay organized during cloning to combine the mutagenic PCR products into the correct groups, and to clone into correct vectors.
And during sequencing preparation, to use the correct primers. Extending from this procedure, the main steps of the protocol could be modified to examine mutation combinations, different selection environments, and other protein systems, in order to answer additional questions concerning the genetic and environmental context-dependence of mutations. After watching this video, you should have a good understanding of the work required to construct a whole-protein saturation mutagenesis library, and to assess the functional effects of each mutation simultaneously, using high-throughput sequencing.
Don't forget that working with ethidium bromide and ultraviolet light can be hazardous and precautions such as gloves, lab coat, and UV shield should always be taken while performing parts of this protocol.
