1. What do you expect the outcome of the transformation experiment with psbC-Syn

Question

1. What do you expect the outcome of the transformation experiment with psbC-Synechocystis to be?

2. Does transformation of psbC-Synechocystis with the recombinant plasmid and the wild type genomic DNA serve the same purpose? Explain.

3. Discuss the purpose and procedure of transformation of psbC-Synechocystis.

used to transform the mutant psbC- strain to revert it to wild type and to restore its ability to grow photoautotrophically Experiment Il Overview E. coli is not the sole bacterium that is amenable to molecular biology. There are many such bacteria. In this lab, one of these, the cyanobacterium Synechocystis sp. PCC6803 is introduced. This cyanobacterium has several advantages that make it a very useful organism for gene manipulations. The two main advantages are: Figure II-1. Schematic model of the photosystem Il complex from cyanobacteria cytoplasm/stroma 1. Natural transformability. In contrast to E. coli and most other bacteria, it 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. D1 CP43 02 CP47 The blobs are polypeptides, the wiggles" with circles at the end represent lipids making up the bilayer 2e Pa of the membrane. Names of polypeptides and cofactors have been indicated 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. 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 (Figure 2). Chi Chl As most prokaryotes, Synechocystis has a double-stranded circular genome. 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. MnMn Mn lumen 12kD 33 kD The doubling time of Synechocystis is 12 hours (versus 20 minutes for E. col), and it takes 7-10 days to get visible colonies of transformants on a plate 2 H20 02 +4 H This experiment consists of two parallel and eventually converging parts, which are distinguished in the experiment outline by the letters-A" and-B". Part-A" of the experiment starts by introducing a mutation in the psbC gene of wild-type Synechocystis. This gene codes for an intrinsic chlorophyll-binding protein CP43 that is essential in the assembly and function of photosystem II (Figure 1) The kanamycin-resistant phenotype of the Synechocystis psbC- mutant will be used for segregation. Complete segregation will be confirmed by performing PCR using primers upstream and downstream of the interrupted part of the psbC gene. After segregation, cells no longer have an active photosystem Il complex and depend on sugar addition for their survival In part "B" of the experiment, part of the wild-type psbC gene will be amplified and will be cloned in a plasmid. The recombinant plasmid is amplified in E. coli, sequenced, and

Explanation / Answer

The cyanobacteria, Synechocystis sp., contains a gene called psbC, which encodes for protein CP43. This is a chlorophyll-binding protein for photosystem II.

Thus, the wild type Synechocystis sp containing psbC genes, is capable of growing without any additional carbon source, as they can produce sugars by photosynthesis.

1. Outcome of transformation:

The transformation involves two parts:

A) Introduction of mutation in psbC gene by creating a mutant type, by homologous recombination with plasmid pKCP43 (containing Kanamycin resistance gene). The mutant has Kanamycin resistance gene, splitting the psbC gene.

Thus, this step will be indicated by non-functioning of psbC gene. Thus, cells will require carbon source in growth medium. Moreover, growth in media with Kanamycin will indicate the mutant cells. These factors will be selection criteria for transformed cell.

B) The second step includes transformation with recombinant plasmid (pUC19 in E. coli cells, in this case). The presence of ?-galactosidase in pUC19, helps in blue-white screening of transformed cells, by using X-gal in medium.

The recombinant plasmid thus obtained, is re-transformed in mutant Synechocystis cells. If transformation occurs the cells will be again able to grow in medium without added carbon source.

Thus, this indicate:

a. The readiness of Synechocystis sp. Genome to integrate by double recombination, and transformability.

b. The role of psbC gene in photosystem and photosynthesis. Also, the ability to grow as autotrophs and heterotrophs.

2. Transformation with wild type DNA would indicate that the Synechocystis sp. Has ability to grow under different condition. As autotroph, due to presence of psbC gene, they can make their own sugar by photosynthesis. Also, in mutant variety, as heterotroph, in presence of additional carbon source.

Transformation with recombinant DNA, proves the natural transformability. As the Synechocystis sp. genome, readily takes up foreign DNA.

3. Procedure and purpose:

1. Transformation of wild types with plasmid pKCP43.

2. Series of gradual steep addition of Kanamycin, in medium, mutants with Kanamycin resistant genes, were conducted to select the transformed cells. The medium also contained added carbon source.

The cells that were initially growing as autotrophic (in wild type). Was also able to grow in presence of carbon source.

3. Extraction of genome from transformant.

4. Transformation with pUC19 containing E. coli cells. The transformant were selected by media with X-gal, by blue to white screening. This indicated natural transformability capacity.

5. The selected cells were purified, and the genome extracted and re-transformed into mutant cells. The mutant cells are again capable of photosynthesis.

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