Part:BBa_K3457041

OtsBA

    This biological part is the sequence containing two CDS, OtsB and OtsA. They can catalyze the dimerization of glucose to produce trehalose. trehalose can help bacteria resist stresses. Here we found that expressing OtsBA may help bacteria survive the freeze-drying process and then the resultant dry bacteria powder can be stored for a long time at room temperature.

Group: QHFZ-China iGEM 2020

Design

Figure 1. The Schematic cartoon of the DNA construct of BBa_K3457041.

    OtsBA is a widely used part, our design was not newfangled (Fig.1). Two restriction enzyme cutting sites were added between OtsB and OtsA, so if you can add some sequence between OtsB and OtsA. This year, the part is used in the composite part BBa_K3457043.

    Many similar parts can be found in this library (https://parts.igem.org/),.

Documentation:

Introduction:

    This year, we tried to introduce a new biopreservation method. We used freeze-drying to make the engineered into dry powder. Then the powder can be stored at room temperature for a long time. This method can make the storage of bacteria get rid of ultra-low temperature freezer, so that it will promote the practical application of engineered bacteria out of laboratory. However, the stresses during freeze-drying and subsequent dry storage, including freeze, dry and vacuum, are lethal to bacteria. We use TDPs to help bacteria survive the situation. We also tested the effect of OtsBA (producing trehalose) for comparison.

Protocol:

    To test the effect of OtsBA, we modified a frequently and widely used vector, pet28a+ and put this part into it (Fig. 2). Then we transformed the plasmid into E. coli BL21 strain.

Figure 2. The Schematic cartoon of the vector.

    Then we used the following protocols to verify its function (Fig. 3):

Figure 3. Experiment protocol.

【Day 1】Induction culture
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
【Day 2】Freeze-dried
(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the experiment.
(2) Use spectrophotometer to measure the OD₆₀₀ of the bacteria solution, OD₆₀₀ = 1 equals to 10⁹ cells. If the OD₆₀₀ value is between 0.1 and 1, There is a linear relationship between OD₆₀₀ and bacterial density. Calculate the volume of bacterial solution for 10⁹ cells by using the formula V = 100 / (OD₆₀₀ × Dilution ratio).
(3) Take out a measured amount of 10⁹ cells and centrifuge it at 8000 rpm for 3 min. Then pour out the supernatant.
(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.
(5) Take off the cover of the tube and put the bacteria into the cold trap. Open the compressor of the lyophilization machine and freeze the shake tube for 2 h at -70℃.
(6) Put the caky bacteria solution into the drying chamber of the lyophilization machine. Open the vacuum pump to dry it in vacuum for 6h at 1 Pa vacuum degree.
(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room temperature.
【Day 3】Room temperature storage
【Day 4】Detect the survival rate
(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.
(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria solution on the LB plate.
(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after several gradient dilutions.
(4) Culture the bacteria overnight at 37℃.
【Day 5】Cell Count
(1) Take out the LB plate and take photos to record experimental results.
(2) Use the automatic cell counting function of Image J to count the colone number on the LB plate, then compare the results between each group.

Results:

    First, by a reporter, sfGFP, we confirmed that the plasmid can normally expressed exogenous proteins in E. coli BL21 strain (Fig. 4).

Figure 4. Via a reporter, sfGFP, the expression function of the modified pet28a vector was verified.

<p style="clear:left;">    Then, by freeze-drying and recover experiment, we tested the protective effect of SAHS 33020. However, in an independent repeat test, we found that compared with the control group (sfGFP), the bacteria expressing OtsBA did not have a better survival rate (Fig. 5).

Figure 5. An independent repeat test of freeze-drying and recovery.

    However, we thought that the result above was not credible. It was a little difficult for us to hold 10 samples for one time, so we thought maybe something was wrong. In some other independent repeat tests, bacteria expressing OtsBA had a better survival rate than control group (Fig. 6).

Figure 6. Trehalose (OtsBA) might protect bacteria during freeze-drying and subsequent dry storage. During this repeat, we used puc57 vector to load this part.

Summary:

    Trehalose (OtsBA) may help the bacteria survive freeze-drying and subsequent dry storage. However, the effect were not steady. Further study should be taken to determine the function of Trehalose (OtsBA).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]