From 2003, there are plenty of iGEM engineered bacteria made but most couldn’t have a chance to be taken out of lab. One of the most important reason is that the product of bacteria is labile, so it’s hard to transport or storage.

Facing this problem, one of the solution is to immobilize or reduce the water content of our engineered bacteria to stabilize these products. The traditional way is drying by using high temperatures, but it will make the marked changes in the physical and chemical properties of the product. Freeze-drying is a better solution to provide a stable bacterial product which combines the benefits of both freezing and drying. So we use this method to make the labile bacterial product controllable and active by vacuum desiccation. Though it still can’t be taken out of lab, we think is a promising option.

We made a equipment as an accessory of East-Wind to check the survival of freeze-dried bacterial expressing BFP. You can get more information about it in our wiki.

Below is our experiment and results of freeze-drying, containing the experiment conditions and protocol.

We used the BioBricks T7 +LUXAB, T7+RFP and BFP transformed into BL21(DE3) to make a prime verification.

I. Experiment conditions

1. Bacterial concentration (OD):
1.1~1.2.

2. Protective Agents:
5%, 10%, 20% skimmed milk; Luria-Bertani broth; 10% sucrose solution; 10% sucrose + Luria-Bertani.

3. Pre-freezing conditions:
We chose three conditions as following:
(1)-20℃ for 2h;
(2)-80℃ for 1h;
(3)Gradient freezing: 4℃ for 30 min, -20℃ for 30 min, -80℃ for 1h.

4. Operational Conditions of Lyophilizer:
Firstly, start the freeze dryer to pre-freeze for 2 hours to allow the chamber temperature to drop to -50℃;
Then, sublimate for 12 hours under vacuum condition which called Primary drying;
Finally, increase shelf temperature to about 30℃ to desorb the residual water under vacuum condition.

5. Resuscitation:
First, add 1mL LB or ultrapure water to the sample;
Then, shaking cultivation for 1h under 37℃;
Last, add 2 ‰ IPTG to induce gene expression.

6. Plate Culture:
Incubate 250μL solutions of bacteria that is diluted by 10000 times for 12 hours in 37℃.

II. Experiment Protocol

1. Measurement of OD:
After shaking cultivation to some concentration, measure OD600 of bacterial solution with ultraviolet spectrophotometer. It should be 1.1~1.2.

2. Centrifugation:
1 mL solution of bacteria, 12000r for 2 min.

3. Resuspension:
Add 500μL protective agents which we used are 5% skimmed milk, 10% skimmed milk, 20% skimmed milk, Luria-Bertani broth, 10% sucrose solution and 10% sucrose + Luria-Bertani to each centrifuge tube and then resuspend them.

4. Pre-freezing:
Put the centrifuge tubes into the refrigerator to freeze in three different condition: gradient freeze(4℃ for 30 min、-20℃ for 30 min、-80℃ for 1h), -20℃ for 2h, -80 ℃ for 1h.

5. Operate the lyophilizer:
Start the lyophilizer to allow the chamber temperature to drop to -50℃.

6. Freeze-drying:
After pre-freezing of the sample, put it into the lyophilizer. Then sublimation for 12 hours under vacuum condition which called Primary drying; Lastly, increase shelf temperature to about 30 ℃ to desorbe the residual water under vacuum condition.

7. Resuscitation:
Dissolve the sample by adding 1mL LB or ultra-pure water. Then use the shaking machine to cultivate for 1h under 37℃. After that, add 2 ‰ IPTG to induce gene expression.

8. Cultured on the plate:
Dilute 10μL solution of bacteria by 10000 times. Add 250μL of the bacteria solution to plate and culture it for 12h.

9. Observe and Analyze the Results.

I. The shape of the sample

skimmed milk


LB+10%sucrose

II. The activation of the bacteria after freezing-drying

1. T7+LUXAB

All the data were the bacteria of OD=1.25. The title is the protective agents, and the first line contains the pre-freezing conditions and resuscitation agents.The values were read by the chemiluminescence instrument.






Another batch of freeze-drying：




Grads means: 4℃ for 30 min, -20℃ for 30 min, and -80℃ for 1h.

2. T7+RFP



The resuscitation solutions are water and LB. We resuscitated, shaked, and induced these bacteria which can express RFP after the induction of IPTG.

The blue part of the chart shows the condition od the bacteria in corresponding location.

III. Cultured on the plate

These pictures are the results of plate culturing. All the plates in pictures are placed in the order of pre-freezing conditions of grads , -20℃ for 2h and -80℃ for 1h (from left to right).


LB


5% skim milk


10% skim milk


20%skim milk


LB+10% sucrose


10% sucrose

2. T7+RFP

From top to bottom, the protective regents are 10% sucrose, 10%skim milk, LB+10% sucrose and LB.
We have three plates for every conduction.



-20℃ for 2h for pre-freezing

From top to bottom, the protective regents are LB, 10% sucrose and LB+10% sucrose.
We have four plates for every conduction.


-80℃ for 1h for pre-freezing

Summarize

1. 20% skim milk,10% sucrose and the LB+10% sucrose as the protective regents are presumably more stable than other concentration skim milk;
2. LB+10% sucrose is presumably the appropriate regent, if you want to test the water without adding LB;
3. In some ways, the activation of the bacteria is positive with the survival number.