By Levi Clancy for Student Reader on
- 5'-Deletion Mutants
- Ames Test
- cDNA Microarray
- Cloning Vectors
- DNA Fingerprinting
- DNA Miniprep
- Gel Shift Assay
- Gene Control in Development: Laboratory Techniques
- Gene Targeting
- Genetic Engineering
- Genetic screen
- In Vitro Nuclear Run-on Experiment
- Interrupted Mating Experiment
- Knockout mutation
- Linkage analysis
- Polymerase Chain Reaction
- Promoter (Transcriptional) (RNA) Fusion
- Reporter Gene
- Restriction Enzymes (Endonucleases )
- Sequence Alignment
- Shotgun sequencing
- Temperature Sensitive Mutant Experiment
- Translational (Protein) Fusion
- Transposon Tagging
Determining whether or not cells can transfer genetic material by direct contact. Lederberg and Tatum began with 2 mutants of E. coli: (A) was Bio-Met-, (B) was Thr-Leu-. When these cells are plated on Bio-Met-Leu-Thr- agar, there will be no growth. When at least 108 of these cells are mixed, and then plated on Bio-Met-Leu-Thr- agar, then there is growth. This could be due to transformation, though, and further experimentation is required. Conjugation is much less efficient than transformation, and you could not use boiled lysates and direct cell-cell contact still might not be required. To test this, Lederberg and Tatum constructed a U-Tube with media and in the middle was a cell-proof filter that was permeable to macromolecules. When these are plated on Bio-Met-Leu-Thr- agar, there is no growth. When the filter is removed, there is growth. As a result, direct cell-cell contact is required.
Transfer is 1-way (donor to recipient)
Not all cells are donors
Recipients become donors
Xsmal genes are rarel transferred
The hypothesis is that donor cells have a fertility factor transferred to recipient cells. The fertility factor is a non-xsm genetic element called the F-factor (renamed F-plasmid).
There is a donor, which shall be called F+. This is the first discovery of a plasmid. The F-plasmid carries genes for pillus formation (tra) and DNA transfer, and an origin of transfer. There are a huge number of naturally occurring plasmids, and they can carry various traits, including: drug resistance, metal ion resistance, virulence factors and metabolic pathways. Figure 10.20 shows the R-100 plasmid.
Hfr has a high frequency of recombination and is integrated into the host xsm. It is constantly being expressed.
The Interrupted Mating Experiment uses conjugation to determine the order of genes.