Team:QHFZ-China/Part Collection

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Part Collection

Introduction

We try to apply for Best Part Collection Award because we measured a series of parts that can protect bacteria from freeze-drying. As far as we found, no one studied the parts in live cells druing freeze-drying. We will gave details in this page and thank you for attention.

Description

    There is no ultra-low temperature freezer for bacteria storage in our daily life, so the practical application of engineered bacteria is limited. 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 store bacteria get rid of ultra-low temperature freezers to promote the practical application of engineered bacteria out of the laboratory. However, the stresses during freeze-drying and subsequent dry storage, including freeze, dry, and vacuum, are lethal to bacteria. We use Tardigrade intrinsically Disordered Protein (TDPs), including Cytosolic-abundant heat soluble protein (CAHS) 89226, CAHS 94205, CAHS 106094, CAHS 107838 and secreted abundant heat soluble protein (SAHS) 33020, to help bacteria survive the situation. We also measured two other parts besides TDPs, which are LEA and OtsBA.

    TDP is a kind of heat soluble protein found from Tardigrade Hypsibius dujardini in 2017 ^[1]. Tardigrade, also called water bear, is a kind of tenacious organism. It can survive extreme environment, such as desiccation, freeze and vacuum. The super capacity of Tardigrade partially owes to TDPs. Thus, we designed to let bacteria express TDPs to help itselves survive the freeze-drying and subsequent dry storage. Before TDPs were found, people used two elements to enhance the stress resistance, LEA and trehalose. Thus, we also tried the effect of the two parts, (1) Late Embryogenesis Abundant (LEA) protein, LEA3 protein, from soybean (Glycine max), and OtsBA (OtsB and OtsA), two enzymes that can dimerize glucose into trehalose.

Overview of the part collection

Table 1. Part Collection

Group	Part Name	Short Description	Inducer	E. coli BL21 (DE3)	E. coli DH5alpha
Basic	BBa_K3457009	CAHS 89226 with a 6X His tag	-	-	-
Basic	BBa_K3457011	CAHS 94205 with a 6X His tag	-	-	-
Basic	BBa_K3457012	CAHS 106094 with a 6X His tag	-	-	-
Basic	BBa_K3457013	CAHS 107838 with a 6X His tag	-	-	-
Basic	BBa_K3457014	SAHS 33020 with a 6X His tag	-	-	-
Basic	BBa_K3457040	LEA with a 6X His tag	-	-	-
Basic	BBa_K3457041	OtsBA	-	-	-
-	-	-	-	-	-
Composite 1st	BBa_K3457032	T7-Olac-RBS-CAHS 89226	2 mM iPTG	Yes	-
Composite 1st	BBa_K3457033	T7-Olac-RBS-CAHS 94205	2 mM iPTG	Yes	-
Composite 1st	BBa_K3457034	T7-Olac-RBS-CAHS 106094	2 mM iPTG	Yes	-
Composite 1st	BBa_K3457035	T7-Olac-RBS-CAHS 107838	2 mM iPTG	Yes	-
Composite 1st	BBa_K3457036	T7-Olac-RBS-SAHS 33020	2 mM iPTG	Yes	-
Composite 1st	BBa_K3457039	T7-Olac-RBS-SAHS 33020-T7-Olac-CAHS 106094	2 mM iPTG	Yes	-
Composite 1st	BBa_K3457042	T7-Olac-RBS-LEA	2 mM iPTG	Yes	-
Composite 1st	BBa_K3457043	T7-Olac-RBS-OtsBA	2 mM iPTG	Yes	-
-	-	-	-	-	-
Composite 2nd	BBa_K3457046	J23100-Olac-RBS-CAHS 106094	2 mM iPTG	Yes	Yes
Composite 2nd	BBa_K3457049	J23107-Olac-RBS-CAHS 106094	2 mM iPTG	Yes	Yes
Composite 2nd	BBa_K3457055	araC-PBAD-CAHS 106094	0.2% L-arabinose	Yes	Yes

Overview of our contribution

(1) CAHS 89226 was not existed in the iGEM library before and we are the first to register it.
(2) For CAHS 107838, it has been registered by others but they did not offer any data. We modified the part and we are the first to add experimental data.
(3) For other basic parts, there exist some data, but none data is about freeze-drying live cells. We modified the parts and we are the first to add data about freeze-drying live cells.
(4) For all parts, we optimized the sequence to make it suitable to be expressed in E. coli. For all parts other than OtsBA, we added a 6x His tag, for easy detection by Western blot and purification by Ni-chelating affinity chromatography. (5) This year, we focused on CAHS 106094 most, so we confirmed its function by many independent experiments.

Design

For the basic parts, we gave CAHS 86226 as an example and most parts are like it.

For the composite parts and the vector we used, most are like the following cartoon.

Protocol

    To test the effect of CAHS 89226, 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):

【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 2 mM iPTG/ 0.2% L-arabinose 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.

Result

    By a reporter, sfGFP, we confirmed the function of the gene circuit in E. coli BL21 (DE3) strain.

    By the parts of group "composite 1st", TDPs can be normally expressed.

    We have a screening of the parts. CAHS 89226, CAHS 94205, CAHS 106094 and LEA enhanced the survival rate than control group (sfGFP).

    We think it is too difficult for us, high school students, to hold so many group at the same time, so there may be something wrong in SAHS 33020, CAHS 107838 and OtsBA. In other independent experiments, they also gave a sruvival rate improvemrnt

SAHS 33020, as well as BBa_K3457039 SAHS 33020 + CAHS 106094:

CAHS 107838:

CAHS OtsBA:

    The above test are all in E. coli BL21 (DE3) strain. However, we also constructed some parts that could be used in other strains. We changed the T7 promoters. J23100, J23107 and arabinose inducible promoter Pbad were used. The following results are collected in E. coli DH5alpha strain.

J23100-CAHS 106094:

araC-Pbad-CAHS 106094:

Summary

    In summary, we construsted and measured many parts that protect bacteria from freeze-drying for the first time. The parts can respectively used in different bacteria strains with different inducers, as well as with different effects.

Reference:

[1] Boothby, T.C., Tapia, H., Brozena, A.H., Piszkiewicz, S., Smith, A.E., Giovannini, I., Rebecchi, L., Pielak, G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. Mol Cell 65, 975-984 e975.

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