Growth curves and when to induce

A growth curve tells you when a non-pathogenic E. coli cloning or expression strain is actually dividing, which is the decision behind adding inducer. Lactose-analogue induction of a lac-type promoter is commonly done in exponential phase because that is when the cells are a working protein factory. Stationary cells are biomass with the factory idling. How the strain is grown and transformed in the first place is set out in bacterial culture and transformation. Keeping the plasmid that carries the promoter is part of plasmid cloning from insert to colony, and the antibiotic in the flask is the subject of antibiotic selection and blue-white screening.

Broth, agar and inducer classes can be specified from the reagents and chemicals catalogue. The surrounding methods sit on the molecular biology pathway. If the open reading frame itself is what you need to specify, the custom gene synthesis reference is an enquiry prompt. Use the quote request for the medium and the inducer specification. A method can be discussed there.

The decision the curve is for

You are choosing a moment on one flask, not a universal clock time. Two cultures inoculated at 9 o'clock can be in different phases by noon if the inoculum size, the aeration or the temperature differed. Induce because the culture is in exponential growth on your instrument, not because a protocol from another laboratory said "three hours". The vector system you are using still has the last word: some promoters are tighter, some proteins are toxic, and some hosts (a T7 expression strain is the usual example of that class) respond harder than a plain cloning host. Follow that system's note. This page only explains why the phase matters.

There is no secret titre here. Yield in milligrams per litre is a result you measure for your protein. It is not a number this article can hand you. What the curve can do is stop the two classic mistakes: inducing so early that the culture never thickens, and inducing so late that you are asking stationary cells to start a new protein programme.

Lag, exponential growth and stationary phase

After you dilute a colony or an overnight into fresh broth, the cells spend a lag rearranging their metabolism. Optical density barely moves. Division is rare. Adding inducer here asks a cell that is still resetting to make your protein on top of that reset. If the product is at all burdensome, the culture can stall while it is still thin. You then harvest a small, stressed population and call the protein "low expressing".

Exponential, or log, phase is the stretch where turbidity rises steadily and the doubling time is roughly constant. Ribosomes are abundant. Amino acids and a carbon source are still available in a rich broth. This is the window in which lac-type induction usually works, because the cell can spare capacity for a plasmid-borne gene. A widely used description of that window is mid-exponential growth. Laboratories adjust the exact moment. A band often quoted for routine E. coli work sits somewhere around the middle of the rise on that laboratory's own spectrophotometer, and many vector sheets gesture at readings in a mid-range band near half an absorbance unit to a bit under one. Those figures are not portable. Map them once, then trust the shape of your curve.

Stationary phase begins when a nutrient runs out or waste products slow division. Turbidity levels off. Cells become smaller, ribosome numbers drop, and stress regulons switch on. The culture looks satisfyingly dense. It is a poor moment to turn a promoter on and expect soluble, folded product. Some of the protein you do get is more likely to pack into inclusion bodies, because folding lags synthesis and the cytoplasm is no longer in balanced growth. Harvesting a culture that was induced in mid-exponential and then allowed a few more hours is a different decision from inducing only after the curve has already flattened.

Optical density is turbidity, measured locally

A spectrophotometer at 600 nanometres reports how much light failed to reach the detector. Cells scatter that light. The reading also moves with cell size, medium colour, bubbles, a scratched cuvette and the blank you set. It is not a colony-forming unit count until your laboratory has related the two for that strain, that medium and that instrument. An overnight that has lysed can stay cloudy. A culture of very small cells can be denser in cell number than the absorbance suggests.

Read in the linear range of your instrument. Above that range the number compresses and you think the culture is still climbing when it has already slowed. Dilute a thick sample into the same broth and correct for the dilution rather than believing an off-scale reading. Blank against sterile medium from the same bottle. A blank of water makes rich broth look like growth before any cells are added.

Shake flasks need air. A flask filled high into the neck becomes oxygen-poor, the curve bends early, and the pH can drift. That early bend is not "this protein is toxic". It is geometry. The Addgene protocol for inoculating a culture shows the ordinary shape of starting a cloning-strain broth from a colony. Volume, temperature and shake speed still belong to your shaker, not to a borrowed sentence.

What early and late induction do to the protein

Induce too early and two things compete. The cells have not yet reached a useful density, so even a healthy specific yield leaves you with little material. And if the product slows growth, the induced culture is overtaken by any cell that failed to respond, or it simply stops. You see a curve that rises and then quits, and a pellet that is mostly unhappy biomass.

Induce too late and the promoter may still fire, but the translation machinery is winding down. Total protein can look weaker on a gel, or it can appear as a strong insoluble band because the folding capacity was already committed. Lowering temperature after induction is a common next experiment for solubility. It does not repair a culture that was induced in deep stationary phase. Change one variable at a time: phase first, then temperature, then how much inducer the vector note suggests. IPTG is the usual gratuitous inducer for lac-type systems. The amount is a property of the promoter and the host. Take it from the vector sheet and confirm it with a small side-by-side, rather than from a number remembered from a different plasmid.

Keep the selective antibiotic in the expression flask unless you have a reasoned exception and a way to show the plasmid is still there. A burdensome induced protein makes plasmid loss more rewarding for the cell. A cloudy culture of plasmid-free daughters will not show your band. That failure belongs with plasmid stability, not with the spectrophotometer.

Where on the curve induction usually belongs

When the curve you plotted is not the culture you have

A blank that was water, a flask volume that changed aeration, or a culture that sat without shaking will give you a confident OD and the wrong phase. If the positive expression control (a plasmid you have induced before in this host) also fails, look at temperature, air and inducer stock before you redesign the gene. If the control induces and yours does not, the phase was probably fine and the open reading frame, the codon set or a mutation is the next place to look. Gels and stains used to see that band are a separate method; reading a protein gel is the companion for how much trust a lane deserves.

Do not induce a culture that smells wrong, has clumped, or came from a plate with mixed morphologies. You would be measuring a contaminant’s growth curve. Dead cells scatter light too. A plateau can mean stationary phase or a culture that has already crashed. A viable count, or a simple restreak, separates those stories when the absorbance has gone ambiguous.

Expression cultures stay inside the institutional rule

The strains in view are K-12-style cloning and expression hosts your institution has already accepted. Scaling a flask is not permission to grow a different organism because the shaker has a free clamp. Inducer stocks and antibiotics are chemicals with their own hazards. Follow the safety note that arrived with them. This page is not medical advice and not a metabolic-engineering recipe. The WHO Laboratory Biosafety Manual is a public reference for the people who set containment. It is not applied to your building by this article.

Waste broth is inactivated by the route the laboratory already uses. Do not pour a live expression culture down a sink because the gel looked empty.

Heat, power cuts and a curve that stopped

In a hot building an incubator set to 37 Celsius can run warmer if the room is already close to that temperature and the compressor is struggling. A culture induced "at 37" may have spent the afternoon at a higher temperature, which is one ordinary way to push a protein into inclusion bodies. Check the flask, not only the set point. A power cut stops the shaker. The culture sits warm and still, oxygen falls, and the exponential rise you were waiting for is over even if the clock says it should continue. When power returns, do not add IPTG to that flask and pretend the phase is unchanged. Cool it, and start again from a colony or from a backup that was still shaking.

An afternoon OD blank can also drift if the spectrophotometer itself is hot. Re-blank. Write the instrument name next to the number so the next person does not copy your absorbance onto a different machine.

What to put on a medium or inducer enquiry

Name the host class, cloning versus expression, the promoter family, rich broth versus a defined base, and the inducer. Say whether you need the antibiotic in the same broth. Ask for composition and grade in writing. The quote request is the place for that specification. A method can be discussed. The custom gene synthesis reference can carry a question about the open reading frame. It does not say that a synthesis is already being performed. Public method collections such as Promega protocols are useful for seeing how suppliers describe expression steps. They are not a growth curve for your shaker.