Difference between revisions of "20.109(S22):M2D4"
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| + | ====Transform CRISPRi system into MG1655 ''E. coli'' cells==== | ||
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| + | During transformation, a plasmid enters a competent bacterium, then replicates and expresses the encoded genes. In a co-transformation, the goal is to transform each bacterial cell with two plasmids that each encode a different set of genes. Following the co-transformation procedure, a mixed population of cells exists as shown in the figure to the right: some cells only contain the plasmid that carries the resistance cassette for antibiotic A (blue cells), some cells only contain the plasmid that encodes the resistance cassette for antibiotic B (red cells), and some cells contain both plasmids (purple cells). Because the agar plate used for selection contains both antibiotic A and antibiotic B, only bacterial cells that harbor both plasmids survive and reproduce to form a colony. | ||
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| + | [[Image:Fa20 M3D3 cotransformation schematic.png|thumb|right|550px|'''Schematic of bacterial co-transformation.''' Bacterial cells that harbor both plasmids (purple cells) are selected for using an agar plate that contain antibiotics.]]In the CRISPRi system, a gene on the psgRNA_target expression plasmid encodes an ampicillin-resistance cassette and a gene on the pdCas9 plasmid encodes a chloramphenicol-resistance cassette. Thus, only a co-transformed bacterium will grow on agar plates containing the antibiotics ampicillin and chloramphenicol. | ||
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| + | Most bacteria do not usually exist in a “transformation ready” state, referred to as competence. Instead bacterial cells are incubated with CaCl<sub>2</sub> to promote competency by making the cells permeable to plasmid DNA uptake. Competent cells are extremely fragile and should be handled gently, specifically the cells should be kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 10<sup>9</sup> transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed. | ||
==Reagents list== | ==Reagents list== | ||
Revision as of 18:45, 24 January 2022
Contents
Introduction
The sgRNA_target sequence that was inserted into the expression plasmid was confirmed using DNA sequencing. The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive process. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s and 2000s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides, or chain-terminating bases. These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA sequence due to the presence of fluorescent dyes, one color linked to each dideoxy-base. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected.
Protocols
Part 1: Participate in Communication Lab workshop
Our communication instructor, Dr. Prerna Bhargava, will join us today for a discussion on preparing a journal club presentation.
Transform CRISPRi system into MG1655 E. coli cells
During transformation, a plasmid enters a competent bacterium, then replicates and expresses the encoded genes. In a co-transformation, the goal is to transform each bacterial cell with two plasmids that each encode a different set of genes. Following the co-transformation procedure, a mixed population of cells exists as shown in the figure to the right: some cells only contain the plasmid that carries the resistance cassette for antibiotic A (blue cells), some cells only contain the plasmid that encodes the resistance cassette for antibiotic B (red cells), and some cells contain both plasmids (purple cells). Because the agar plate used for selection contains both antibiotic A and antibiotic B, only bacterial cells that harbor both plasmids survive and reproduce to form a colony.
Most bacteria do not usually exist in a “transformation ready” state, referred to as competence. Instead bacterial cells are incubated with CaCl2 to promote competency by making the cells permeable to plasmid DNA uptake. Competent cells are extremely fragile and should be handled gently, specifically the cells should be kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.
Reagents list
Next day: Prepare for induction of CRISPRi system
