Description

Various engineered bacteria showed useful functions. However, the storage of these bacteria usually requires -80℃ refrigerators, which substantially limited their usages in daily life. Therefore, we decided to utilize TDPs from tardigrades and freeze-drying to supply a new storage method, dry powder storage at room temperature. We confirmed that certain TDPs can maintain the survival rate of E. coli. Then, we further optimized the method. We hope the method would promote the practical application of engineered bacteria.

Promotion video

Inspiration

In the project of our team last year, QHFZ-China 2019, the team members designed a portable household uric acid detector, whose core is a kind of genetically engineered bacteria. However, because the goal of this project is to develop an easy method to facilitate gout patients, it is also important for the bacteria to be accessed by everyone. Unfortunately, during communication, we knew that last year, the team members were unable to find a way to store the bacteria at normal people’s home. The reason is that in lab, bacteria are stored at ultralow temperature so that their metabolism pauses. However, to normal people, they do not have necessary equipment.

The project of our team (QHFZ-China) lasr year (2019). It is convenient to gout patients, but the requirement of -80℃ freezer is a difficulty. QHFZ-China 2020 will try to solve the problem.

Based on the above , we figured that many other teams may experience the same issue of not being able to utilize their project because of storage issues. After we conducted interviews with 10 iGEM teams, we realized that the storage issues are actually quite common. Thus, to develop a convenient biopreservation method to store bacteria became our goal for the project.

8 out of 10 teams thought a new bioprotection method wihout low temperature would be quite helpful for the practical application of their peojects.

Besides, nowadays, bacteria need to be transported at a low temperature, -80℃ or at least 4℃. However, during our interview with Jiajia Liu and Miatta Momoh, we found the cold chain was lacking. And if the bacteria can get out of the limit of cold chain, the use of engineered bacteria will be much more convenient.

Cold chain is lacking, expensive and easy to go wrong.

Current limitations

Currently, in laboratories, biopreservation mainly needs ultralow temperature (-80℃) refrigerators, which are not accessible to normal people. Scientists need to first add in glycerin then mix it with bacterial solution before putting it into the freezer for storage. Moreover, when bacteria are thawed, the glycerin should be removed, so that professional machines like pipettes or centrifuges need to be used. All the things above makes it impossible to be used in daily life.

Of course, there are some other methods for bacteria storage, though they are rarely used. As far as we know, they include "Agar Slant Cultures", "Paraffin Preservation" and "Sand Preservation". However, they have shortcomings respectively, such as short shelf life, affecting the functions of bacteria, complicated operation. For example, "Sand Preservation" uses sterile sand to store the spores of bacteria. However, it is complicated to make sand-spore mix; the sand is easy to cause pollution and affects the observation; most importantly, the method is only suitable for the bacteria that can produce spores.

Hence, it is crucial to find a method of biopreservation suitable popularization among normal people.

Brainstorming

We began to search for a method to pause the metabolism of life instead of ultralow temperature. From our textbook, we learnt that if the water content of a cell reduces, the cell's metabolism slows down. Hence, we decided to make the bacteria into dry powder. Indeed, after information research, we found desiccation is truly a workable plan. Simply drying needs a long time (about 7 days), which is unfit. During that time, the sate of cells could gradually become worse. As a result, we decided to use freeze-drying (lyophilization) to make dry bacteria powder, because freeze-drying is much more faster and can maintain the initial state of freeze-dried material. Then the dry powder can be stored at room temperature without any professional equipment, which is obviously convenient.

Survival stresses

However, freeze-drying and subsequent dry biopreservation bring stresses that affect the survival rate. That are freeze, desiccation and vacuum. So we must use some protective measures. As far as we have researched, nowadays, people most take protective measures outside the cells, such as optimizing the components of the protective solution (freeze-dried liquid) and the freeze-drying protocol. Nevertheless, the survival rate is usually less than 10%, which should be enhanced. In all, we decide to further enhanced the survival rate with another idea —— adding some resistance genes inside the cells.

Tardigrade (water bear)

Tardigrade is a kind of miraculous organisms. It can survive extreme environments, like freeze, desiccation, vacuum, extreme temperature, high pressure and radiation. It was found in 1773 and is famous for strong vitality. Hence, we hope to find some inspiration from Tardigrade.

Tardigrade (water bear), a kind of hardy organism.

TDPs (Tardigrade Disorder Proteins)

In 2017, scientists found that tardigrades produce a kind of unique proteins, Tardigrade intrinsically Disordered Proteins (TDPs). By experimental verification, they found that the strong vitality of tardigrades at least partially owes to TDPs, especially resistance to desiccation ^[1]. At the same time, they found heterologous expression of TDPs helps E. coli BL21 (DE3) strain and yeast survive desiccation ^[1]. Then iGEM teamTUDelft 2017 and XMU-China 2018 proved that TDPs protects certain proteins during freeze-drying _{[2, 3]}. During our interview with Jiajia Liu, we knew that among the three stresses during freeze-drying ( freeze, desiccation and vacuum), desiccation might be the worst one to bacteria. As a result, we speculated that TDPs could protect bacteria from freeze-drying and subsequent storage in the form of dry powder.

TDPs protect bacteria from desiccation and purified proteins from lyophilization.^{[1, 2]}

Our design

We decided to make bacteria dry powder. The powder can be stored at room temperature for a long time. When using it, you only need to add some medium. We will use a bottle to hold the medium in our product. We used freeze-drying to achieve the powder and TDPs to improve the survival rate and cell activity.

Our imagined product: a kit containing dry bacteria powder and medium. Refrigerators are no longer needed.

As far as we know, people have found TDPs protect bacteria from drying, and protect proteins from freeze-drying. But no one studied if TDPs could protect living cells during freeze-drying. So our data would give insight to TDPs.

In addition, all data of TDPs came from E. coli BL21 (DE3) strain and all data were qualitative. What's more, no one has ever combined different TDPs to see if they had synergistic effect. As a result, besides BL21 (DE3) strain, we would try to get some data from other strains. We would also get some quantitative data and combine different TDPs to test the synergistic effect.

More information

Indeed, the low temperature biopreservation method also has other problems. For example, when we interviewed an iGEMer, Yikai Bao, he told us that last year, their -80℃ refrigerator broke down and they suffered great loss; when we visited TIDE pharmaceuticals company, Mr. Lu told us that -80℃ cannot make the metabolism of bacteria pause completely, so the storage time is limited and the cell state would be influenced. Temperature lower than about 140℃ can stop the metabolism. However, liquid nitrogen should be used to achieve the temperature, which is troublesome. In all, the method should be improved to bring a better security and stability to bacteria in store.

References

[1] G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Mol Cell 65, 975-984 e975.
[2] iGEM Team TUDelft 2017, https://2017.igem.org/Team:TUDelft
[3] iGEM Team XMU-China 2018, https://2018.igem.org/Team:XMU-China