Intro The synthesis of altered proteins with new binding sited, designed to target specific groups of molecules, is one of the major tasks of modern protein engineering. Significant efforts in this area were highlighted by limitations of natural proteins in both biomedical and analytical applications…
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Using these two techniques it is possible to synthesize a protein that will bind any desired target. As recent studies suggest, it is possible to add random peptide sequences into loops of ?-lactamase subsequently establishing the catalytic properties of the produced ?-lactamase derivatives. The same authors highlighted the fact that there is no correlation between tolerance to insertion and tolerance to mutagenesis. A turn between two ?-strands next to the active site was found to be inactive in random mutagenesis but demonstrated the opposite in insertions. The present work consists of three elements. Initially it is creating a construct (cloning a mutated gene into an expression vector) ?-lactamase a. using traditional cloning methods (overlapping PCR for mutagenesis, digestion, ligation). Then move on to Protein- Prep- expressing and isolating mutated ?-lactamase a, transformation of construct into competent cells b and protein purification by GFC and IEC before, finally, move on to investigating the effects of mutation on the functionality of ?-lactamase a. Activity assay of mutants compared to those of the WT enzyme A Procedure Week 1: PCR- Primer Design/PCR Mutagenesis Two sterile 0.2 ml PCR tubes were loaded with 5 µL PFU buffer, 3 µL dNSO, 2.5 µL template, 0.5 µL PFU, 26.5 µL H2O each. Also, one tube was loaded with 5 µL Reverse Primer and 5 µL Forward Primer Mutant while the other was loaded with 5 µL Forward Primer and 5 µL Reverse Primer Mutant. 23 cycles of PCR were used to generate the required amount of the DNA sequence of interest. Denaturation, annealing, and elongation represent one cycle of PCR. The first minute of DNA generation was conducted at 950C the second at 500C. The temperature for the following three minutes was raised to 720C with subsequent 10 minutes of elongation at 720C before finally cooling down to 40C affording the crude product. Week 2: PCR Fragment Purification and Restriction Digest A. The crude product produced on the previous stage was loaded into the wells of 0.4 % agarose gel, the first run was conducted. All bends were cut and 330 µL QG buffer was added. The mixture was heat till the gel dissolved completely after that transferred to the column and span for 2 minutes. 500 µL QG buffer was added and spinning was continued for extra three minutes. 30 µL EB buffer was added to dissolve DNA and spinning was continued for 2 minutes. In this way DNA was pulled through. B. To generate the required amount of DNA PCR was conducted. Each of the two sterile 0.2 ml PCR tubes were loaded with 5 µL PFU buffer, 5 µL Forward Primers, 5 µL Reverse Primers, 2.5 µLdNTP, 0.5 µL pfu, µL H2O. Also, one tube was additionally loaded with 5 µL AB DNA (Forward mutant) while the other 5 µL CD DNA (reverse mutant). On the next day the first tube was loaded with DNA 30 µL, Eco R1 buffer 4 µL, Eco R1 - 1 µL, Hind III- 1 µL, H2O- 4 µL and the second with 4 µL vector, Eco R1 buffer 4 µL, Eco R1- 1 µL, Hind III- 1 µL, H2O- 4 µL. Both tubes were left at 370C overnight. Week 3: Restriction Fragment Purification/Ligation/Agar Plate Preparation The gel run was initiated following purification of the previously generated DNA samples. DNA concentration was measured and was found to be 5 µL into 500 µL. The following ligation was conducted. The ratio PCR/Vector was 3/1 Week 4: DNA Transformation/
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The author of the paper explains that β-lactamase enzymes are responsible for a high degree of bacterial resistance to β-lactam antibiotics, having mutated and evolved with the development of new and stronger antibiotics. Thus, hundreds of unique bacterial lactamases have been described.
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