- Methods to obtain amino acid alphabet
- In past few years, directed evolution emerged as the most efficient and effective protein engineering methods.
- It has many application in basic as well in biotechnology, synthetic organic chemistry.
This methods used for the improvement of some traits like:
- Protein activity
- Folding affinity
- Inter alia
- Selectivity (enantio-, stereo-, and regioselectivity).
- Stability (pH, temperature, and organic solvents).
- It consists of repetitive cycles of mutagenesis, screening or selection or expression of random or focused gene of the fittest variant.
- Various methods are employed to create diversity in which the most common one being error prone repair (epPCR), Saturation Mutagenesis and homologous recombination based methods like DNA shuffling.
- Although, epPCR and DNA shuffling are the most common methods in directed evolution, from these methods semirational approaches have emerged because the same or better results are obtained with less effort.
- From these one of the methods are there which are depends on the Iterative Saturation Mutagenesis (ISM), in which careful designing of the library is crucial for the success.
- Activity can be increased by substrate scope and stereoselectivity and regioselectivity, amino acids lining are grouped and randomized near the substrate binding pocket using the Combinatorial Active-site Saturation Test (CAST).
Strategies have been developed for selecting the optimal AAAs
- Bioinformatics and computational method
- Random methods
- Rational method
Suitable database are used to obtain the nucleotide sequence of interest, e.g., GenBank.
- Cloning or assembling of target gene into a suitable vector.
- In ultra-pure water or suitable buffer, good quality oligonucleotides are suspended at a concentration of 2-5 μM.
- PCR Master mixture prepared which includes buffer, dNTPs, and polymerases.
- Restriction enzyme is added such as DpnI.
- Expression host: E. coli BL21 (DE3) Gold.
- Ingredients of SOC medium: 20 g/L tryptone, 10 mM MgSO4, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, and 5 g/L yeast extract.
- Media was then sterilized properly by autoclaving, 20 mM glucose added using 2M of sterile stock solution.
- Luria Broth (LB) medium and LB agar plates prepared.
- On the basis of plasmid suitable antibiotic stocks solution(s) was added.
- Plasmid maxiprep, midiprep, and/or miniprep kit.
- Gradient thermocycler suitable for 96-well microtiter plates (MTPs).
- High-throughput (HT) DNA electrophoresis.
- Incubator with shaker for MTPs.
- Incubator with shaker for Eppendorf tubes.
- Centrifuge for MTPs and Eppendorf tubes.
- Spectrophotometer for determining DNA concentration.
- Multichannel pipettes (8 or 12 channels).
- Standard plastic ware (PCR tubes, 1.5 and 2.0 mL plastic reaction tubes, 15 and 50 mL falcon tubes, petri dishes of 10 and 15 cm diameter).
- 96-Well MTPs for PCR.
- Agarose gels for HT DNA electrophoresis.
- Standard 14 mL round-bottom tubes for cell culturing.
- 2.2 mL deep-well 96-well MTPs used with paper or metal lids.
- 6-/12-Well multidish plates with flat-bottom well design.
- Glass beads of ca. 3 mm diameter.
HT DNA plasmid extraction and sequencing in MTP format is provided by a company.
Software and Servers
- Programs used for designing of primers in HT format:
- Deep scan is one of the program which is currently in use that allows introduction of degenerate or non-degenerate codons in the primers which are partially overlapped. In contrast, Quik Change mutagenesis which used for gene segments or complete genes.
- AAscan is a technique which was primarily utilized for the finding of optimal primers for the alanine scanning.
- MegaWHOP is a method which based on the method MegaPrimer.
- Software for alignment and processing of the files of multiple DNA sequencing.
- Software for the measurement of base peak heights from DNA chromatograms.
- To calculate library screening effort an online program applied.
Stages of this protocol are:
- Library Design in Silico
- Choose optimal system
- Define target region according to budget allocation
- Design oligos
- Order a few test oligos
- Optimisation conditions for library creation
- PCR with test oligos
- Template digestion, transformation, and cell plating optimization.
- DNA sequencing
- Evaluation of preliminary libraries
- Determine QQC and Q values
- Q values should be less than 0.6
- Library creation
- High-throughput mutagenesis PCR
- Transformation and sequencing
- Identity missing mutation
- Order oligos for library completion
- Library completion
- HT site directed mutagenesis for obtaining missing variants.
- HT transforming in MTP
- HT DNA sequencing in MTP
- Library evaluation
- Construction of master plate
- Investing sequence function relationships
- Screening of master plate.
- Construction and analysis of mutability landscape using excel and or Matlab.
- Construction of combinatorial library based hotspot
Reference and Sources