1. What specific genetic manipulation does Synechocystis readily allow that E. c
ID: 31437 • Letter: 1
Question
1. What specific genetic manipulation does Synechocystis readily allow that E. coli does not?
2. For transformation of Synechocystis, why do you first plate your transformant mixture
on a filter on a BG - 11 plate with glucose, and the next day the TA transfers the filter
with cells to a plate that contains kanamycin (rather than plating directly on kanamycin)?
3. What is the product of the psbC gene? What is its function?
4. In the PCR reaction, what is the role of each of the PCR reagents: primers, dNTPs,
Mg2+, and Taq polymerase?
5. What are the considerations in determining the number of cycles needed for the PCR
reaction?
Explanation / Answer
1)
There are 2 reasons.
1. Natural transformability. In contrast to E. coli and several other bacteria, Synechocystis spontaneously takes up foreign DNA that is present in the growth medium, and can integrate it into its genome through a double homologous recombination event. Double homologous recombination is appropriate to introduce gene interruptions or deletions using a construct with two regions of sequence identity with the cyanobacterial genome.
2. The ability to grow in many different conditions. It can grow photoautotrophically utilizing its photosynthetic system to produce sugars from CO2 and water, using light for energy. It also can grow photoheterotrophically utilizing a reduced carbon source in the growth medium. This characteristic helps Synechocystis survive under different growth conditions, and cope with a mutation in its photosynthetic system.
2)
The doubling time of Synechocystis takes about 12 hours (versus 20 minutes for E. coli), and it takes 7-10 days to get visible colonies of transformants on a plate. As most other prokaryotes, Synechocystis has a double-stranded circular genome (dsDNA). However, in contrast to many other prokaryotes, it carries multiple (6-12) copies of the genome in a single cell. As upon transformation only a single genome copy in the cell may be altered, wild-type and mutant genomes are slowly sorted out (segregated) upon repeated cell divisions. A Synechocystis mutant means that all genome copies carry the same mutation, and that the wild-type genome copies are absent. Segregation of mutants is achieved through a steep increase in the antibiotic concentration in the growth medium. Cells that carry the mutation in every copy of the genome are at a selective advantage to cope with high concentrations of the antibiotic.
3)
The mutation is introduced by interrupting the psbC gene by a kanamycin resistance gene via double-homologous recombination with the pKCP43 plasmid. This plasmid contains part of the psbC gene interrupted by a kanamycin resistance gene. This gene psbC codes for an intrinsic chlorophyll-binding protein CP43 that is essential in the assembly and function of photosystem II.
4)
Polymerase chain reaction (PCR) enables the amplification of any DNA sequence (producing millions or even billions of copies) in just a few hours. An important feature of PCR is that the DNA segment chosen to be amplified does not need to be separated from the rest of the genomic DNA prior to initiating the amplification procedure. However, once amplified, the segment can readily be separated from the bulk of DNA (which is not amplified) by gel electrophoresis. PCR requires buffer, primers, and nucleotides (dNTPs). Keep dNTP on ice at all times. After adding dNTP to your sample, place samples on ice. Wearing gloves when handling the reagents can eliminate the possibility of contaminating the samples by proteases, DNase, or an alternate template.
PCR primer is required to synthesis of new DNA strand. Taq polymerase is the enzyme carriesout the reaction. The dNTPs added extend the DNA strand and Mg2+ ions required for the psbC gene amplification.
5)
Numbering of the psbC locus starts at 1 and terminates at 1418. In the PCR reaction in a day the amplification of the sequence between 662 and 2819 is observed and this means that amplification occurs at the 3' end of the gene and the adjacent (flanking) region.
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