Term
| Term Project: Make E. Coli glow using the lux operon from V. fischeri |
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Definition
| Glowing Bacteria V. fischeri -- isolate chDNA -- repurify chDNA -- digest chDNA and plasmid DNA for shotgun cloning of lux operon -- ligate chDNA into plasmid DNA to create V. fischeri Genomic library -- Transform E. Coli with Plasmid library -- screen E. Coli for plasmids containing lux operon (glowing colonies) -- re-isolate plasmids from lux positive E. Coli for mapping |
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Term
| Gram Negative Bacteria V. fischeri |
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Definition
| Chromosome, Pilus, Cell Membrane, Cell Wall, Capsule (slime layer), plasmid, flagellum, inclusion, ribosomes, pilus (fimbria) |
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Definition
96% of all deep sea marine organisms have a symbiotic relationship with bacteria
exact benefit to fish unknown (defense, communication, attracting prey)
bacteria (nice cushy, nutrient rich environment) |
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Definition
Production of light requires a lot of energy and oxygen (bacteria won't glow unless their environment allows optimal growht)
Threshold cell density required (process by which a colonizing bacteria knows that "critical mass" has been achieved is called QUORUM SENSING) |
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Definition
| Bacteria produce a diffusible compound (N-Acyl homoserine lactone), the more bacteria, the more inducer. Once the levels are high enough, bacteria knows that nutrient levels are good and energy can then be expended |
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Definition
| Enzyme that catalyzes the light emitting reaction (heterodimer of alpha and beta subunits), mixed function oxidase**, reaction requires the simultaneous oxidation of Flavin mononucleotide and a long chain aldehyde |
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Term
| Three enzymes needed to generate the long chain aldehyde that Luciferase will oxidize |
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Definition
1. Acyl Transferase
2. Acyl - Protein Synthetase
3. Acyl- Reductase |
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Term
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Definition
| removes fatty acids from their normal biosynthetic pathway for use in this reaction |
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Term
| Acyl - Protein synthetase |
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Definition
"activates" the fatty acid to form R-CO-AMP
ATP DEPENDENT! |
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Term
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Definition
Reduces the activated fatty acid to form the necessary aldehyde
NADPH DEPENDENT |
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Term
| Biochemistry of Bioluminescence |
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Definition
FMNH2 + O2 + R-CHO --> FMN + R-COOH + H2O + LIGHT |
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Definition
| Encodes for the autoinducer |
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Definition
| Encodes for the transcriptional activator that binds to the inducer |
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Definition
| encodes for the acyl-reductase enzyme |
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Definition
| encodes for the acyl transferase enzyme |
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Definition
| encodes for alpha subunit of luciferase - has the active site of the enzyme |
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Definition
| encodes for the beta subunit of luciferase - absolutely necessary for the activity of the enzyme |
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Definition
| encodes for the acyl-protein synthetase enzyme |
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Definition
| genetic regulatory system in which genes coding for functionally related proteins are clusted along the DNA. This feature allwos protein synthesis to be controlled coodinately in response to the needs of the cell |
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Term
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Definition
| in a bacteria, it is a large circular piece of DNA. for our project, the chromosomal DNA comes from V. fischeri and we will be isolating the lux operon from this |
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Term
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Definition
| enzymatic reactions that cut DNA into smaller pieces. Mediated by enzymes that will digest the DNA at only specific sequences |
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Definition
| small circular DNA, used to move DNA from one organism into a second, vector |
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Definition
| enzymatic reaction that will "paste" two pieces of DNA together. Pieces can be from the same organisms or two different sources |
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Definition
| term for what is produced when DNA from two different sources (chDNA + plasmid DNA) |
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Definition
| is the practice of randomly digesting a large piece of DNA to smaller pieces that can be ligated into plasmids for transport to other organisms |
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Term
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Definition
| collection of cloned DNA fragments from a genome. Each part of the genome is represented int he library, the library can be screened for the presence of a sequence of interest (in our project the genome is from V. fischeri and the sequence of interest is the lux operon) |
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Term
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Definition
| a bacteria housing a recombinant plasmid that contains apiece of genomic dna, it doesn't have to contain the gene you're looking for |
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Term
| TES buffer - Tris/EDTA/NaCl |
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Definition
| Tris/NaCl: helps keep pH stable and isotonic |
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Term
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Definition
| chelates (sequesters) divalent cations like Mg 2+, aids the lysozyme removing Mg from LPS layer, inhibits DNases |
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Definition
| enzyme that destroys the cell wall |
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Definition
| digests proteins: stable, more efficient in SDS |
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Term
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Definition
strong ionic detergent:
dissolves cell membrane and denatures protein |
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Term
| Phenol, Phenol: chloroform |
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Definition
| removes lipids, protein and polysaccharaides: clean up |
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Term
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Definition
| helps precipitate DNA by charge shielding |
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Definition
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Term
| Adding two volumes of cold EtOH |
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Definition
isopropanol can be used but results in more protein contamination
When using isopropanol, you use less of it which reduces overall product (advantage if your tube has limited capacity) |
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Term
| Factors affecting nucleic acid precipitations |
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Definition
1. Cations for Charge Shielding
2. Temperature during the precipitation step
3. Concentration of the nucleic acids
4. Time and speed of Centrifugation |
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Term
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Definition
There must be at least .2M of a monovalent cation present to shield the high negative charge of the phosphate in nucleic acids
Without shielding, intrastrand repulsion is too great, and there will be no aggregation |
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Term
| Most common salt for routine DNA isolations |
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Definition
| Sodium Acetate, Sodium chloride may be used if the sample contains/will contain SDS (allows detergent to remain soluble in ethanol solution) |
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Term
| Temperature During Precipitation |
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Definition
| it was commonly thought that precipitation needed to be at lower temperatures, but the recovery is actually better at 0 c (on ice) or at room temperature |
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Term
| Concentration of nucleic acids |
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Definition
precipitations occur effectively within 10 minutes when the nucleic acid concentration is greater that .5 microgram/mL
if the concentration is less than .5 microgram/mL, incubation time must be increased (from 30 minutes to overnight) |
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Definition
nucleic acids are usually collected at 12000 - 14000 x g between 4 C and room temperature
ten minutes is long enough if the concentration is high (> microgram/mL)
if the concentration is low, centrifuge for 30 -60 min OR increase the g force |
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