In this page, we will introduce the designing of our TDPs gene circuit and lyophilization process. More detailed information can be found in proof of concept page. We also give a concisely introduction about the background of our project.
In 2017, a study published on Molecular Cell reported that the TDPs (tardigrade disordered protein) have the ability to improve the resistance of bacteria under desiccation environment. Additionally, in 2019, team QHFZ-China designed a bacterial sensor that can detect uric acid, which greatly benefit gout patients as well as normal people. However, through the communication with QHFZ-China 2019, we realized the usage of their bacteria is limited by the preservation method. Therefore, we wanted to mainly focus on this issue.
During the Human Practice we conducted in this summer, we found that besides QHFZ-China 2019, there are still many other iGEM teams and individuals who face the same issue. We found that the issues all range in the following aspects:
(1) For engineered bacteria, the room temperature storage method and practical applications in normal life are limited by the requirement of professional equipment.
(2) The lack of cold-chain transportation as well as its cost should be improved.
(3) The production of lyophilization products and the survival rate of bacteria after lyophilization process all have rooms for improvement.
Based on the information above, we believe that it is necessary for us to provide a new storage method to achieve a higher survival rate and easier storage for these bacteria. Therefore, we planned to use lyophilization to produce the dry powder of bacteria and also added the TDPs as the protectant for bacteria during freeze-drying.
Design Of TDPs Expression Gene Circuit
We engineered E.coli cells to express TDPs during freeze-drying with the idea of synthetic biology. The gene of TDPs is the core component of the circuit. TDP is a type of protein that originates from the tardigrade, which have resistance to desiccation. The Olac will repress the expression of downstream genes, so we will add iPTG to get rid of this repression and induce the expression of TDPs. By changing the strength of RBS and promoter, we can control the TDPs to be expressed at a suitable level. All these parts mainly exist on the pet28a-modification vector. While doing our experiments, we mainly transformed this vector into E.coli BL21 (DE3) strain. Moreover, to confirm whether our TDPs can have a wide adaptation, we also transformed it into E.coli DH5alpha strain and transferred our TDPs into another vector called pYB1a. To test whether the TDP is expressed, we used the SDS-Page technique.
Design Of Lyophilization
To confirm whether the TDPs can really protect the bacteria during lyophilization, we came up with the following process and the detailed protocol is shown on Experiments page. Additionally, we realized that the ways to pause the metabolism of bacteria include freezing and drying. Taking the suggestions from our human practice, we use the 3% glucose as the protectant of bacteria. There are also some procedures that we took during the process of lyophilization. For instance, we conducted 2 hours of freezing and 6 hours of drying to allow the water molecules in the cells to be sublimated. After the end of the lyophilization process, we placed the dry powder produced inside silicon for 24 hours of room temperature storage. The reason for this storage method is to make sure that the powder can remain in the “cake” state, which we learned was the optimal state of our powder from our human practices activities.
We found out that when using T7 promoter to express TDPs, the protective effect of the individual TDP on the bacteria can still be improved. Therefore, we conducted the quantitative experiments and replaced the T7 promoter with J23100, J23107 and J23109. Through these methods, we were able to precisely control the amount of proteins expressed in our experiments. At the same time, we realized that as the expression rate of the proteins increased, the effectiveness on survival rate also increased simultaneously. To verify that our proteins also function in other E.coli strains, we converted the pet28a-modification vector containing the TDPs into the DH5alpha strain of E.coli. Additionally, when we added the TDPs onto the PYB1a vector, the proteins also displayed the protection effect it displayed on other strains of bacteria. All of these could prove the continuous optimization we conducted onto our experiments. At the same time, it also displayed the universality of our method of storage and our proteins.
In order to decrease the damaging effects of the proteins on our bacteria, we designed a degradation circuit for the protein to "self-destruct" once it achieved its purpose. We added the PDT tag to the C-terminal of the protein. Through the addition of mf-lon, the PDT tag can degrade the TDPs that we have originally placed in the bacteria. We displayed our degradation process through the method of mathematical models. From the results of modeling, we could infer that our degradation circuit will successfully avoid the stresses that over-expression of TDP will add onto the bacteria while simultaneously decreasing the effects of TDP on the original functions of the engineered bacteria. Through mathematical calculations, we will be able to produce the most suitable bacteria dry powders based on the requirements made by the customers, achieving the practical implementation of our project.