OSU Microbiology
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Seminars & Schedules
Seminars & Schedules

Study of Bacterial Gene

Study of bacterial gene expression heavily relies on E. coli-based in vivo and in vitro systems with RNA polymerase at their core. Although methods utilizing RNA polymerase assembled from individually expressed subunits or single-subunit expression vectors have met with considerable success, our experience indicates that co-overexpression vectors, featuring most or all RNA polymerase subunits have substantial advantages, particularly in efficiency of assembly of some mutants, deemed “defective” based on in vitro assembly attempts (see for example Artsimovitch et al, Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions, JBC, 2003, 278(14):12344-55). Here we present the latest co-overexpression vector for E. coli RNA polymerase, pVS10, and a sample protocol for purification of the enzyme produced using this vector (although it should be compatible with any classical RNA polymerase purification protocols as well).

pVS10 vector contains ORFs for 4 subunits, comprising E. coli core RNA polymerase rpoA (a), rpoB (b), rpoC (b’), and rpoZ (w). This plasmid has been successfully used as a platform for genetic manipulations of rpo genes and production of the wild-type as well as a number of mutant enzymes. It has been tested for expression in BL21(DE3), XJb(DE3), and DH5a(DE3) cell lines using both IPTG– and autoinduction (e.g. Overnight Express, Novagen) protocols. Yield varies with the toxicity of the construction, the identity of the strain and the induction conditions, usually within 1-10 mg/L of culture.

Induction and growth conditions:

The autoinduction using the Overnight Express (Novagen) reagents was carried out according to the manufacturer’s protocol with modifications to preculture preparation. BL21(DE3) cells transformed with pVS10 plasmid were inoculated from the frozen stock into 30 ml of LB medium (Miller) supplemented with ampicillin at 100 mg/L in 125 ml flask and grown with agitation at 37C till OD600 =0.6, when 0.6 ml of this culture were used to inoculate 600 ml of LB medium (Miller) supplemented with carbenicillin at 100 mg/L in 2 L flask, supplemented with Overnight Express reagents per Novagen protocol. The culture was incubated at 32C with agitation for 18 hours, and cells were harvested by centrifugation (4C, 5000 g, 10 min).

Lysis and Ni-affinity chromatography:

Cell pellet was frozen (-80C) and thawed (on ice), cells were resuspended in 50 ml of lysis buffer (500 mM NaCl, 50 mM Tris-HCl (pH 6.9), 5% glycerol, 0.2 mM b-ME) supplemented with 1X Complete EDTA-free protease inhibitors cocktail (Roche), and 1 mg/ml lysozyme. Suspension was incubated on ice for 60 min with occasional swirling, supplemented with Tween 20 to 0.2% and sonicated briefly to disrupt the cells. Extract was cleared from cell debris by centrifugation (27 000 g, 15 min, 4C). Cleared extract was combined with 4 ml of 50 % Ni-NTA slurry in lysis buffer and incubated with agitation for 30 min at 4C. The slurry was poured into a disposable gravity flow column (Bio-Rad) and drained. The column was washed with 20 volumes (40 ml) of lysis buffer, and 10 volumes of heparin column loading buffer (50 mM Tris-HCl, pH 6.9, 5 % glycerol, 0.5 mM EDTA, 1 mM b-ME) supplemented with 10 mM imidazole. Elution was carried out by adding 1 ml aliquotes of heparin column loading buffer, supplemented with 250 mM imidazole. Fractions with more than 0.2 mg/ml of protein (by Bradford) were analyzed using SDS-PAGE, the ones containing RNAP were pooled and if needed concentrated to total volume of 4-5 ml using Amicon Ultra 100K concentrators (Millipore).

Heparin-affinity chromatography:

RNAP-containing pool of fractions was loaded onto a 5 ml Heparin HiTrap column (Amersham) at 0.5 ml/min using low-pressure LC system (AKTAPrime, GE) (can use Heparin 16/10 columns for larger scale preps, load at 1 ml/min).

Column loaded with polymerase preparation was washed with 5 column volumes (25 ml) of loading buffer, elution was carried out using linear gradient of NaCl in loading buffer : 0-1.5 M NaCl over 120 ml at 1 ml/min. Peak fractions were collected, pooled, and dialyzed against loading buffer (alternatively the ionic strength can be reduced by several cycles of concentration-dilution with loading buffer using Amicon Ultra 100K concentrators).

Ion-exchange chromatography:

Polymerase fractions collected in previous step were loaded onto 6ml ResourceQ column (GE) using the same system at 0.5 ml/min, washing and elution were carried out as described for Heparin Hi-Trap column. Peak fractions were analyzed by SDS-PAGE, pooled, and dialyzed against the DNA-agarose loading buffer (10 mM Tris-HCl (pH 7.9), 0.1 mM Na-EDTA, 0.2 mM b-ME, 5% glycerol).

DNA-affinity chromatography:

Polymerase fractions collected in previous step were loaded onto 5 ml DNA-Agarose HiTrap column (GE) using AKTAPrime LC system at 0.2 ml/min. Loaded column was washed with 4 volumes (20 ml) of DNA-agarose loading buffer, and purified core RNAP was eluted using linear gradient of NaCl in loading buffer 0-1.5 M over 100 ml at 0.2 ml/min. Core RNAP containing fractions were analyzed by SDS-PAGE, pooled and dialyzed against RNAP storage buffer.

Seminars & Schedules

I. HUMAN RELATIONSHIPS

What do people desire in a mate?

Do we differ substantially from our ancestors?

What impact has western civilization had on our selection process?

Are human males and females driven by mating practices that insure survival of the fittest?

Are men selected by women because they have a particular reproductive advantage, i.e., plumage like a peacock? Do females really select their mate and what are the criteria?

What criteria do males and females use for mate selection?

Desires in a mate evolved by natural selection.

Our ancestors struggled to keep warm by the fire, hunted for meat for their kin, gathered nuts, berries, herbs, avoided dangerous animals and hostile humans.

If we selected a mate that failed to deliver these resources, survival would be tenuous and reproduction would be at risk.

The asset of having a mate who was able to provide abundant resources, protected us and our children and devoted time and energy to the family is obvious.

As a result of the powerful survival and reproductive advantages that were reaped on those of our ancestors who chose a mate wisely. Clear desires in a mate evolved and we carry those desires with us today.

 

  1. Men’s desires:

Ancestral man: needed women of high fertility and needed to insure paternity of offspring.

Mating with women of high fertility insured high reproductive success. But fertility could not be determined by our ancient ancestors.

Two cues were used: physical cues to youth and health. Younger women were more reproductive than older women.

Men evolved desires that focused on physical appearance in general and youth in particular.

Evidence from 37 cultures, over 10,000 individuals supports this. Men placed a greater premium on physical attractiveness than do women.

Men universally desire a younger wife. Men also desire women with a low waist to hip ratio (small waist relative to hips) which is a key indicator of fertility and symmetrical body features as evidence for good health.

  Facial features such as clear, smooth skin, eyes, lustrous hair, full lips, absence of sores or lesions are universally regarded as attractive.

Selecting fertile women would do little good if the paternity was compromised, i.e., females mated with several males. Women are 100% sure that their child is their own. Male paternity is always less than 100%.

The value people place on chastity is highly variable, ranging from indispensable in China to irrelevant in Sweden and the Netherlands.

In 62% of the cultures, i.e., U.S. and Europe, men place fidelity as the #1 desired characteristic in a potential spouse.

 

 

B.Women’s Desires:

Heavy parental investment, 9 months gestation, years of breast feeding. Women constitute an exceptionally valuable asset of man.

Parental investment is a critical factor in sexual selection, i.e., the sex that invests more in offspring is selected to be highly choosy about mates.

Ancestral women selected mates with resources and who showed a commitment to invest those resources in them and their children.

Women in all continents, all political systems, all racial, religious backgrounds, place more value on good financial prospects.

Overall, women value financial resources in a mate about 100 percent more than men do, i.e., 2x as much.

There are cultural variations. Japanese women value good financial prospects 150% more than Japanese men. Women from the Netherlands value that criteria less than do women from the rest of the world.

Women also valued ambition, industriousness, older age and social status.

Also physical qualities as good health, size, strength, athletic prowess: cues that signal delivery of resources over the long run and protection of herself and her children. Also dependability and emotional maturity.

 

C.Common desires:

Kindness in mate, intelligence, love and mutual attraction.

 

D.Mate selection

Desires evolved by natural selection

Ancestral needs: food, warmth, protection, fertility

Male desires: fertility, paternity (faithful, caring)

Female desires: financial prospects (provider); family values (protector)

  1. Love at First Sight

Symmetrical sense of balance is attractive, i.e. how males and females view each others features.

 

F.Pheromones

Chemicals released by one animal that cause a behavioral change in another

Vomeronasal organ: (VNO)

  1. -Small chemosensory organ in nose

Detects chemical signals that mediate sexual and territorial behavior

Human:

Male VNO responds to estrogens

Females VNO responds to androgens

Females: Synchronization of menstrual cycles

More then 100 genes code for receptors

Different receptors mediate different behavioral responses

Example: Sexual arousal, aggression

Signals bypass brain higher cognitive centers and stimulate subconcious responses that we are not aware of..