Autoinduction media initial trials

Rationale: will substitute the expensive and cold-chain reliable IPTG with low-cost lactose-based products.

Lactose is cheaper than IPTG (SIGMA: IPTG £55/g meaning 13£/L of media considering an induction at 1mM, alpha-lactose: £36/Kg meaning £0.072/L of media considering 0.2% concentration used in autoinduction media. From public retailers 1kg/lactose can be found sold below £10. Whey or milk are £0.8/L (fresh semiskimmed milk from sainsbury) and we would use 40ml/L, meaning £0.032/L ). Lactose is available from either chemical suppliers (e.g. SIGMA) or pharmacies and e-commerce retailers (e.g. amazon, e-bay).

Recipe for Autoinducible media is based on the simplified version from Imperial College here

Na2HPO4 6g

KH2PO4 3g

Triptone 20g

Yeast Extract 5g

NaCl 5g

50% glycerol 12ml

10% glucose 5ml

5% lactose 40ml

Prepare 4xYT (add Na2HPO4 6g KH2PO4 3g Triptone 20g Yeast Extract 5g NaCl 5g in 0.5L) and sterilize in 1L bottle by autoclave.

Autoclave 1L of double distilled water

Prepare the solutions 50% glycerol 10%(w/v) glucose 5%(w/v) lactose and filter sterilize each solution separately.

Add to the 0.5L 4xYT sterile media 12ml of 50% glycerol 5ml of 10% glucose 40ml of 5% lactose.

Bring to 1L volume with sterile water

Cow milk contains around 5% lactose. In cheese preparations the majority of lactose will end up in the whey. Both milk and whey can be considered a 5% solution of lactose and therefore they can be added to the media as the recipe above.

Materials: milk (can be whole, skimmed or semi-skimmed), around 2 lemons

Equipment: small soucepan, colander, gauze (can be any piece of cotton fabric with a fine mesh)

Bring the milk to simmer

Let it cool down

Add the juice of 1 lemon and stir

wait for 10-15 mins

remove the cheese curds from the whey with the small colander

repeat the procedure bringing the whey to simmer (in this way you'll remove more curds)

filter the remaining whey with the cloth (you'll still have some very fine curd pieces left)

Bring the whey back in tha lab and adjust the pH to 7 with NaOH (adding lemon made the pH to decrease)

Filter sterilize the pH adjusted whey (it will be hard to filter)

you can store the filtered whey at room temperature or in the freezer

Note: with the curds you have created an eatable fresh cheese.

Note: If you don't have lemons, any acid would do the job e.g vinegar

Comparison of protein induction power between IPTG and lactose from both a chemical supplier (SIGMA) and home-made whey.

Method:

Day1

Late afternoon: grow an overnight culture (10ml) of OBL1 strain (BL21DE3 E.coli strain transformed with pOBL1, for the expression of OpenVent controlled by lac operator)

Autoclave 100ml of LB media in 500ml flask.

Prepare 100 ml of autoinducible media with lactose from sigma (cat nr . 61345)

Prepare 100ml of autoinducible media with home made whey

Day2

Inoculate the media 1:200 with the OBL1 overnight culture (0.5ml overnight culture in 100ml of media)

follow the growth every 1 h

Induce the LB culture with IPTG in the exponential phase

Growth was performed in in baffled flasks, shaking at 220 rpm, temperature at 37C. Note that LB final OD reaches 5.17 while autoinduction media reaches 7.97 (lactose) and 8.40 (whey). This is probably due to the fact that the autoinduction media is contains more nutrients per liter when compared with the LB media (LB media: Yeast extract 5 g/L, Tryptone 10g/L, NaCl 10g/L).

Samples were taken to measure OD600 and protein expression on SDS-PAGE.

LB culture has been induced at OD 1.1 (a bit late exponential phase in this case)

OpenVent is expressed in autoinducible media between 6 and 10h from initial inoculum. IPTG shows expression after 2h from induction, and it is clear that offers more control over time of expression.

Can we decrease the time to induction in the autoinducible media?

E.g. decreasing the concentration of glucose?

To answer to this question I've set up 5 autoinducible cultures with 0, 25, 50, 75, 100% glucose (where 100% glucose is the concentration of glucose in the autoinducible media recipe)

For each culture I've harvested samples every two hours from when they reached OD 0.5 (equivalent to 3h in the experiment above), and prepared samples for SDS page. Note that the OD600 was very similar for all the cultures at each time point.

There is no time shift in the expression pattern. This is probably due to other factors that overcome the absence of glucose (e.g. AA and rich media available and oxygentation as explained Studier, F. William. "Protein production by auto-induction in high-density shaking cultures." Protein expression and purification 41.1 (2005): 207-234.) (If there is no difference, it might be good to not add glucose at all?)

Since preparing whey is laborious, it would be interesting to see if milk would work as well.

Price of 1L of semi-skimmed milk, fresh (pasturized, note that pasturization process does not kill spores) is 80p, I would need to add 40ml in 1L of media, meaning the cost will be £0.032/L of media with no preparation needed

Since pasturized milk is not sterile, two autoinduced media were prepared. One containin fresh milk (semiskimmed and pasturized, from retailer) and the another one containing fresh milk that was boiled 10 min at 85C (to avoid caramelizing the sugars).

Autoinducible media prepared as above, but adding directly milk to the culture. The media with milk (either fresh or boiled) has a starting OD600 of 4.

Samples were collected at 4, 6, 8h from the inoculum ( as above: 1/200 in 100ml media, incubated in 500ml buffled flasks, shaken at 220rpm at 37C). The cultures with fresh or boiled milk reached OD 6.3 and 7.5 after 8h, respectively.