BIO 219 FINAL

Project overview purposes

1st —> create a complete A. Fishcheri genomic libraru that will include making e.coli glow by transformation with the lux operon

2nd —> quantify gene expression changes in the transformed e. Coli using gadA gene

Alivibrio fishcheri

A gram negative, rod shaped bacteria found living in symbiosis with a diverse array of marine organisms. It's chromosomal DNA (chDNA) contains the lux operon, which codes for proteins that are necessary for bioluminescence.

Gram-negative bacteria

Bacteria that have complex cell walls with less peptidoglycan but with an external lipopolysaccharide (LPS) layer. Very toxic and hard to treat. They stain very lightly (pink) in Gram stain. They are typically more resistant to antibiotics than Gram-positive bacteria. Examples: A. Fishcheri and E. Coli.

symbiotic relationship between A. fishcheri organism and the creatures that house it

bacterial organisms receive energy in the form of glucose and amino acids from the host

2 examples of marine organisms that take part in symbiotic relationship

angler fish and hawaiin bobtail squid

Operon

A unit of genetic function common in bacteria and phages, consisting of coordinately regulated clusters of genes coding for proteins that are clustered along the DNA. This allows for protein synthesis to be controlled coordinately in response to the needs of the cell.

describe lux operon

responsible for bioluminescence phenomenon. 8.5 kb long. luxR gene: encodes for transcriptional activator which binds to AHL. luxI: encodes for the AHL synthase. the AHL and transcriptional activator bind between luxR and luxI to initiate bi-directional transcription. the gene is transcribed as polycistronic mRNA.

Chromosomal DNA

DNA found in chromosomes but the term is often used to describe the large circular loop of DNA found in bacteria. For the project, chDNA comes from A. Fishcheri and we isolated the lux operon from this.

Restriction digestion

enzymatic reactions that cut DNA into smaller pieces. Mediated by enzymes that will digest the DNA at only specific sequences

Bioluminescence

the production of light by means of a chemical reaction in an organism. 96% of all deep sea marine organisms have a symbiotic relationship with bacteria.

Example of bioluminescence

Angler fish and bacteria.

Fish benefit —> communication, attracting prey, hiding from predators, attracting a mate

Bacteria benefit —> reliable food source such as glucose and amino acids

biochemical reaction that causes bioluminescence

* FMNH2 + O2 + R -CHO →FMN+R-COOH+H20+LIGHT
* -catalyzed by luciferase

Quorum sensing

Regulation of the lux operon's gene expression in response to changes in cell-population density. bacteria are sending changes in growth

Auto-induction

The AHL (N-Acyl homoserine lactone) produced by the luxI gene is able to freely diffuse across the cell membrane into the extracellular environment. When the bacterial population increases, more AHL is produced, increasing the concentration in the extracellular environment. At a high enough concentration, the AHL diffuses back into the cells to cause changes in gene expression.How bacteria senses growth;

AHL

Bacteria releases N-Acyl homoserine lactone (AHL), which diffuses across the cell membrane and into the extra cellular environment.

sdiA

e. coli luxR homolog

Can E-Coli use quorum sensing?

Though E. coli cannot produce its own AHL, it can use quorum sensing by detecting the AHL produced by other organisms. The AHL produced by other organisms can regulate portions of the genome, specifically in regard to the upregulation of the gadA gene.

GADAX operon

The gadAX operon is a portion of the genome found in E. coli as part of the AR2 (acid resistance system 2). It contains the gadA gene which encodes for a glutamate decarboxylase, aiding in acid resistance. SdiA detects the AHL molecules to induce upregulation of the gadAX operon.

2 samples from experiment

The experimental sample, taken from a glowing colony, containing E. coli and pGEM lux+. The other sample, the negative control, contains E. coli and pGEM lux-. (She might also be asking for gadA and 16S rRNA?)

As the density of the bacteria increases

More of the inducer is released into the extra cellular environment

When a critical concentration of the inducer is present in the extra cellular environment

The inducer diffuses back into the cells and interacts with the lux operon, which results in gene expression

Substrates that fit into active site of luceriferase

FMNH2 and a long chain fatty aldehyde

What is the enzyme that catalyzes the light emitting reaction?

Luceriferase

structure of the lux operon

Composed of luxR, luxI, luxC, luxD, luxA, LuxB, and luxE.

LuxI

autoinducer synthase, encodes for autoinducer

LuxR

A DNA binding transcriptional activator that needs high concentrations of autoinducer; encodes for the transcriptional activator that binds the auto inducer

luxC

encodes for the Acyl-reductase enzyme

luxD

encodes for the acyl-transferase enzyme

LuxA

encodes for the alpha subunit of luciferase - has the active site for enzyme

luxB

encodes for the beta subunit of luciferase - absolutely necessary for the activity of enzyme

LuxE

Encodes for acrylic-protein synthetase enzyme

acyl transferase (luxD)

removes fatty acids from their normal biosynthetic pathway for use in this reaction

Acyl-Protein synthetase (luxE)

"activates" the fatty acid to form R-CO-AMP

\-ATP dependent

\

Acyl-reductase (luxC)

Reduces the activated fatty acid to form the necessary aldehyde

\-NADPH dependent

Resuspension Buffer (Buffer ATL)

contains sodium dodecyl sulphate (SDS) a strong anionic detergent that can solubilize the membrane proteins. The detergent disrupts the lipid bilayer of gram-negative cells and brings the proteins into solution as protein-lipid-detergent complexes. The phospholipids in the membrane are also solubilized by the detergent. SDS helps release the DNA binding proteins by denaturing them and binding both membrane and non-membrane proteins as monomers. Small soap bubbles also appear

Proteinase K

Reagent for dna isolation; enzyme that will aid in the release of nucleic acids while deactivating nucleases (enzymes that degrade nucleic acids) present. The addition of this enzyme degrades these nucleases and protects the nucleic acids from nuclease attack. In addition, this enzyme is stable over a wide pH range and is well suited for use in DNA extraction.

RNase A

Ribonuclease that is used to degrade RNA that is present in the sample. It cleaves 3' side of phosphodiester bonds after pyrimidine nucleotides in single stranded RNA. DNA remains intact bc it doesn't have the 2'OH group required by this ribonuclease for forming the cyclic intermediate cleavage product.

Solubilízame membrane proteins with a mild detergent

This disrupts the lipid belayer and brings the proteins into solution as protein-lipid-detergent complexes. The phospholipids in the membrane are also solubilized by the detergent.

Lysis Buffer (Buffer AL)

contains a chaotropic (chaos-forming) salt called guanidinium chloride, which is a reference to the ability to disrupt the regular hydrogen bond structures in water. In vivo, nucleic acids are covered by a hydrate shell consisting of water molecules that maintain the solubility of DNA in aqueous solutions. When guanidinium chloride is added to the nucleic acid, the hydrate shell and hydrophobic interactions between neighboring stacks of base pairs are disrupted. This sets up the conditions for the DNA to selectively bind to the silica resin in the transfer tube. Chaotropic salts also further denature residual proteins.

Ethanol (binding)

will enhance and influence the binding of nucleic acids to silica by aiding the lysis buffer in creating a more hydrophobic solution.

Wash buffer 1 (AW1)

Has a low amount of chaotropic salt that binds to and removes the proteins and colored contaminants

Wash Buffer 2 (AW2)

Contains ethanol to remove the salts added from the other buffer, aw1

DNA grade water

Free of salts, DNases, and proteases which allows for the re-hydration and re-maturing of the DNA, causing it to lose affinity for the silica (pH 5.4-7 at 25C)

Binding - silica column w/ h20

SDS dissociates in presence of chaotropic salts in the lysis buffer and sodium ions for a cation bridge to bind DNA to

Spectrophotometric analysis of DNA

DNA absorbs in the UV range, at a wavelength of about 260 nanometers (nm). It absorbs at this wavelength due to the nitrogenous bases (adenine, guanine, cytosine and thymine) of DNA

What has higher absorbance in spectrophotometry analysis?

SsDNA due to lack of shielding from the phosphate backbone

Beer-Lambert Law

law stating that intensity of color change is directly proportional to the concentration of an analyte in a solution

A = ecl

(Absorbance) = (extinction coeff)(conc. Of substance)(light path length, in cm)

Absorption at 260 nm

Absorbance max for nucleic acids (DNA and RNA)

Absorption at 280 nm

Absorbance maximum for proteins due to amino acids tryptophan, tyrosine, and cysteine

Absorption at 230 nm

Nucleic acids minimum absorbance. High readings indicate contamination w/ organic compounds and salts like SDS

260/280 ratio

—PROTEIN/RNA contamination

260/280 ratio for pure DNA

Between 1.8 and 2.0

High 260/280 ratio

\>2.0 —\> RNA contamination

Low 260/280 ratio

260/230 ratio

Purity w/ respect to organic compound and salt contamination

260/230 ratio purity

\
should be between 2.0-2.2

\-sample is pure of any sample association w/ this ratio.Should be between 2.0 - 2.2

Low 260/230 ratio

spectrophotometry

When white light passes through a prism it separates into each of its wavelengths which pass through a thin slit selected for by the machine. The detector sits on the other side of the slit with the sample in between. Material in the sample will absorb the light. Any remaining light passes through and is picked up by the detector.

Shotgun cloning

Randomly digesting a large piece of DNA to smaller pieces that can then be lighted into plasmids for transport to other organisms

How long is the lux operon?

8.5 kb

Size of a plasmid vector

Must be large enough to hold workable quantities of foreign DNA but also small enough to be retained by the host. Also small enough to be distinguishable from the host chDNA

High copy number of a plasmid vector

50 to 100 per cell

origin of replication of plasmid vector

Must be able to copy often and independently of host DNA. Must also be recognizable by the hosts replication machinery

multiple cloning site (MCS)

A region of DNA containing sequences for many restriction enzymes. Unique on vector.

\-cutting w/ enzymes in MCS = linearization, not fragmentation

selectable marker in plasmid vector

For the uptake of vector and foreign DNA. Often used to resist antibiotic, b-gal (blue/white colonies), and green fluorescent proteins

RNA polymerase promoter sequences in a good plasmid vector

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\-mRNA can be made off of inserted DNA

\-it's usually near the MCS

\-often 1 on each side so directionality is NOT an issue

Plasmid vector of choice

\-pGEM

\-gem = Gemini = 2 promotors

pGEM3zf(+)

\-MCS: disrupts LacZa gene

\-Selectable marker: amp resistance + laczA for insertion of foreign DNA

\-RNA polymerase sequences: 2 promotors: T7 and SP6

\-frank cloning site so inserted gene can be expressed irrespective of orientation

restriction endonuclease def

enzyme that can recognize and cleave specific DNA sequences. Used naturally by bacteria to present infection and also cuts viral DNA.

\-named for organism of origin

\-each recognizes a specific sequence of DNA (palindromic/symmetrical sequences)

Volume example

If given certain concentration of chDNA like 2 mg/mL and we need volume of chDNA to get 1.5 mg:

\
(1.5 mg)/(2 mg/mL) = 0.75 mL of stock chDNA

Agarose gel

\-gel matrix used for electrophoresis

\-range between 0.7% - 2%

Agarose gels w/ higher % agarose

Smaller pores

Agarose gel w/ smaller %

Larger pores

0.7% agarose gel

Good separation (resolution) of larger DNA fragments (5 - 10 kb)

2% agarose gel

Good resolution for smaller DNA fragments (0.2 - 1 kb)

Nucleic acid stain for electrophoresis

Gel red: consists of ethidium subunits that are bridged by a linear spacer.

\-fluorophore, and properties are identical to ethidium bromide

\-when exposed to ultraviolet light, it will fluoresce w/ an orange color that strongly intensifies after binding to DNA

\-marketed as less toxic

blunt ends

Restriction fragments with no overlapping ends and that never combine with another type of DNA

Sticky ends

the uneven ends of a double-stranded DNA molecule that has been cut with a restriction enzyme

restriction endonucleases rules

\-must recognize 6 base pair sequences

\-since there are 4 bases in dna, this means that any specific 6 bp sequence will show up every 4 thousand base pairs (4 kb)

\-problem bc lux operon is 8.5 kb

Sal I enzyme recognition site

GTCGAC

\-high GC content: 67%

A. Fishcheri GC content

Low in genome, only 40% of bases are GC. ensures more precise cutting at about 9 kb

Low genomic GC and high GC in enzyme's recognition site \=

Sal I will cut A. Fishcheri LESS frequently than expected

In our digestion, average length is 9 kb

\-operon remains intact

\-fewer clones to screen

Complete digestion of chDNA on restriction gel

First 3 lanes spread from 10 kb to 2.5 kb

Bacteriophage lambda control

Genome: 48.5 kb linear dsDNA

\-0.5 kb fragment should show up in the digested lambda lane

Ligation reaction

Reaction that forms recombinant DNA molecules by covalentes bonding 2 restriction fragments w/ compatible ends

\-creates covalent linkage to repair DNA strands w/ sticky ends of DNA fragments

T4 ligase

type of DNA ligase(enzyme) from bacteriophage T4 and which is capable of ligating blunt ends

\-uses ATP as energy

\-magnesium (Mg 2+) as cofactor

\-requires free 3'OH group on 1 fragment and free 5'PO4^(3-) group on other fragment

\-will synthesize an ester linkage between the 2 groups

ester linkage

linkage that occurs between fragments as a result of ligation

Ligation rxn outcome 1 (most likely to occur)

Plasmid vector could ligate back on itself w/ no genomic DNA fragments

Ligation rxn outcome 2 (2nd most likely to occur)

Multiple fragments could ligate to each other and become circularized

Ligation rxn 3 (3rd most likely to occur)

Plasmid could accept multiple fragments

Ligation rxn 4 (least likely to occur)

Plasmid accepts one fragment

Best molar ratio of Insert:Vector (I:V)

3:1

\-best ligation/transformation rate bc there are more available free ends of chDNA to reduce likelihood of plasmid self-ligation while not being so much that many chDNA fragments ligate together

It's better to set up multiple ratios to improve overall chances of good ligation:

1:1, 2:1, 3:1, 4:1

Good ligation gel

L1:1_0 —> digested pGEM3zf(+) @ 3- 4 kb

L1:1_ligated —> A. Fishcheri chDNA @ 10 kb and self-ligated pGEM3zf(+) @ around 2kb

Transformation

Genetic alteration of an organism brought about by the incorporation of foreign DNA into cells

\-not necessarily integrating foreign DNA into host genome, it can remain separate

horizontal gene transfer

the acquisition of foreign DNA by one of three methods: transformation, transduction, and conjugation.

transduction

bacteriophage/vector to insert foreign DNA

conjugation

transfer of DNA from another organism through direct contact

Transformation of E. Coli

Could force E. Coli to become competent w/:

\-ice-cold calcium chloride

\-heat shock

\-return to ice to restore membrane rigidity