Trends in Biotechnology 110512 – Ch 3c Vectors and other ways of transformation

To selectively kill cells and select for successfully transformed bacterial cells. Exact copies of the original bacterial colonies are made by replica plating from the original master plate. A sterile cloth pad is placed on the plate, so cells stick to it. The pad is pressed onto other plates, creating exact replicas of the original plate. Antibiotics in the media in the new plates will kill any bacteria that do not have the recombinant plasmid inside of them. The original plate is then examined to see which bacterial colonies were successfully transformed.

Fig. 3.7 Selection for recombinant cells after transformation.

Replica plating

Question: A gene is put into pBR322 plasmids using Sal 1. Which of these colonies have the new gene?

pBR322 was made so we could select colonies that lost antibiotic resistance. But other vectors can be made which show loss of color. The vectors pUC18 and pUC19 (often called pUC18/pUC19 together) have a lacZ’ gene. They can make part of the -galactosidase protein.

Together they can make a complete -galactosidase protein. α-complementation The vectors pUC18 and pUC19 can make part of the -galactosidase protein (the first 146 amino acids). These vectors are put into a bacterium with the last part of the -galactosidase protein. Together they can make a complete -galactosidase protein.

Together they can make a complete -galactosidase protein. This is called α-complementation. When the -galactosidase enzyme is made it can hydrolyze lactose into glucose and galactose. The enzyme can also change a chemical called X-gal. This chemical will become blue.

Fig. 3.8 Restriction map of pUC18/pUC19 showing the ampicillin resistance gene, the origin of replication, and the multiple cloning site within the lacZ’ gene.

Fig. 3.9 The selection of bacterial colonies containing a recombinant vector (pCU vector + DNA insert) by the selection of white colonies.

Bacteriophage can be engineered into a cloning vector. They kill bacteria by two pathways: Lytic pathway (Figure 3.10): Bacteriophage DNA inserted into the bacteria, phage DNA and proteins are made, assembled, and burst out of the bacteria, causing the bacteria to burst (called “lysis”). Recombinant phage DNA can be inserted into bacteria on a bacterial plate. The bacteria die, forming clear areas called “plaques” where the recombinant DNA can be isolated. Lysogenic pathway (Figure 3.11): The bacteriophage genome is integrated into the bacterial DNA, replicating along with the bacterial cell genome. Engineered to only work when recombinant DNA and viral particles are packaged in a test tube and placed on bacterial plates to infect bacteria. Usually hold DNA up to twenty kilobases (kb) in length.

Fig. 3.10 The lytic life cycle of a bacteriophage. Recombinant phage DNA can be inserted into bacteria on a bacterial plate. The bacteria die, forming clear areas called “plaques” where the recombinant DNA can be isolated. Fig. 3.10 The lytic life cycle of a bacteriophage.

Fig. 3.11 The cloning of human DNA restriction fragments (● and ▲) into a bacteriophage vector.

Can hold pieces of DNA between thirty-five and forty-five kilobases. Cosmids. A small plasmid that contains the cos (cohesive termini) sites from phage DNA, a plasmid origin of replication, and genes for antibiotic resistance. Packaged into a bacteriophage protein coat, but the plasmid replicates like a plasmid instead of phage DNA once it is in the bacteria. Can hold pieces of DNA between thirty-five and forty-five kilobases.

Yeast Artificial Chromosomes (YACs). Useful for eukaryotic molecular studies, and contains the following components: A centromere allows the chromosome to be transferred to the daughter cells during cell division. A telomere at the end of the yeast chromosome ensures that the end is correctly replicated and protects against degradation. An autonomously replicating sequence (ARS) that consists of specific DNA sequences that allows the molecule to replicate. A marker gene that provides a way to detect an inserted DNA fragment. Useful to clone large DNA fragments, between 200 and 1500 kilobases.

Example of a Yeast Artificial Chromosome (YAC).

Bacterial Artificial Chromosomes (BACs). Synthetic vectors that have been used to clone very large fragments of eukaryotic chromosomes, between 100 to 300 kilobases. Used in the analysis of large portions of complex genomes, whole genes, and constructing maps of genomes. Created using a small plasmid, called the F (fertility) factor, along with cloning sites and selectable markers. The F factor allows the vector to carry larger pieces of DNA, up to 25% of the size of the bacterial chromosome.

oriS, repE – F for plasmid replication and regulation of copy number. parA and parB for partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC. A selectable marker for antibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection. T7 & Sp6 phage promoters for transcription of inserted genes. Example of a Bacterial Artificial Chromosome (BAC).

Plant Cloning Vectors. Used for purposes such as resistance to disease, pests, and herbicides; improving crop quality and yield; improving nutritional quality; and increasing the life of foods. Most commonly used plant vectors are the tobacco mosaic virus and the Ti plasmid from the soil bacterium Agrobacterium tumefaciens.

Tobacco mosaic virus (TMV) is a positive-sense single stranded RNA virus that infects plants. The infection causes patterns (mottling and discoloration) on the leaves. TMV was the first virus to be discovered.

Agrobacterium tumefaciens and the Ti plasmid: Agrobacterium tumefaciens causes tumor in plants, caused by T-DNA (transferred DNA), located in the Ti plasmid, and contains eight genes that integrate into the plant genome. Engineered Ti plasmids lack the tumor-causing genes, but have the genes required to integrate the DNA of interest into the plant genome. The plasmid is inserted into a plant embryo either by soaking seeds with recombinant A. tumefaciens bacteria, or by inserting the Ti plasmid into cells, which will give rise to the entire plant. Selectable marker genes allow selection of only the plant cells with the plasmid DNA.

Fig. 3.12 The use of Agrobacterium tumefaciens to transfer DNA into plants.

Video: Ti plasmid http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Mammalian Cell Vectors. There are several: Simian virus 40 (SV40) — a small DNA tumor virus, could only hold a small piece of DNA and caused only transient (temporary) expression of the inserted DNA. Retrovirus — a single-stranded RNA virus that contains a gene for the enzyme reverse transcriptase to create double-stranded DNA from RNA template, so that the DNA can integrate into the host cell’s genome. It needs to infect actively dividing cells. Adenovirus — a double-stranded DNA virus that can infect many types of host cells with high efficiency, with a low chance for causing disease. It does not have to infect actively dividing cells.

Mammalian cells are used because bacteria are not able to produce complex eukaryotic proteins that are modified by processes such as glycosylation, or if the mRNA needs to be processed after transcription.

Cell Transformation. Many different cells can have DNA inserted into them, “transformation.” Some bacteria are naturally competent to take in new DNA.

Electroporation If a cell has a cell wall, such as plants, algae, or fungi, enzymes break down the walls. A short electrical pulse opens up pores in the cell membrane, allowing DNA to enter in what is called “electroporation.” The cell wall reforms and cell division starts.

Gene Guns Plant cells can also be transformed by biolistics. Small gold or tungsten particles covered in DNA are shot into cells or tissue at high velocity. Cell walls do not have to be removed.

Fig. 3.13 Particle gun used for transforming cells by biolistics.

An animation of a gene gun can be found at: http://www. hort. purdue

Animal cells can be transformed by electroporation or DNA precipitation with a calcium phosphate solution. Viruses can also be used.

Microinjection of DNA in the cell nucleus, such as an animal egg, is needed to introduce DNA into an entire animal. The DNA integrates into the animal chromosomes, the egg is implanted, and the animal is born with the desired traits.

Fig. 3.14 Microinjection of DNA into pronucleus of a fertilized animal egg.