Difference between revisions of "Part:BBa K3457043"

Revision as of 07:58, 4 October 2020 (view source) Registry (Talk | contribs)		Latest revision as of 00:50, 27 October 2020 (view source) Leaves (Talk | contribs)
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	<partinfo>BBa_K3457043 short</partinfo>		<partinfo>BBa_K3457043 short</partinfo>
−	a	+	<p>&nbsp;&nbsp;&nbsp;&nbsp;This biological part is the expression sequence of 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. </p>
		+
		+	===Contribution===
		+	<h2><b>Group: QHFZ-China iGEM 2020</b></h2>
		+	<h3><b>Author: Yixian Yang</b></h3>
		+	<h3><b>Design</b></h3>
		+	[[File:T--QHFZ-China--ots-11.png|400px|thumb|left|Figure 1. The Schematic cartoon of the DNA construct of BBa_K3457043.]]
		+
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;This part should be composed of a T7 promoter, RBS B0034, CDS of CAHS OtsBA [https://parts.igem.org/Part:BBa_K3457041 BBa_K3457041], and a T7 terminator. However, we added another terminator B0015 before the T7 terminator. The reason is that if the T7 promoter is changed into other promoters, such as J23100, the corresponding RNA polymerase changed from T7 RNAP to endogenous RNAP of <i>E. coli</i>, which can be stopped by terminator B0015. In other words, the added terminator made the part easier to be modified. In the cartoon (Fig. 1), there are two points around the T7 promoter. They are two <i>Bsa</i>I cutting site, which can be used to change the T7 promoter by Golden Gate technique.</p>
		+
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;Many similar parts can be found in this library (https://parts.igem.org/),.</p>
		+
		+	<h3><b>Documentation:</b></h3>
		+	<h4><b>Introduction: </b></h4>
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;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.</p>
		+
		+	<h4><b>Protocol: </b></h4>
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;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 <i>E. coli</i> BL21 strain. </p>
		+
		+	[[File:T--QHFZ-China--ots-12.png|400px|thumb|left|Figure 2. The Schematic cartoon of the vector.]]
		+
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;Then we used the following protocols to verify its function (Fig. 3): </p>
		+
		+	[[File:T--QHFZ-China--freeze-dry protocol.jpg|400px|thumb|left|Figure 3. Experiment protocol.]]
		+
		+	<p style="clear:left;">
		+	【Day 1】Induction culture<br>
		+	(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. <br>
		+	(2) Add 2 mM 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.<br>
		+	【Day 2】Freeze-dried<br>
		+	(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the experiment.<br>
		+	(2) Use spectrophotometer to measure the OD<sub>600</sub> of the bacteria solution, OD<sub>600</sub> = 1 equals to 10<sup>9</sup> cells. If the OD<sub>600</sub> value is between 0.1 and 1, There is a linear relationship between OD<sub>600</sub> and bacterial density. Calculate the volume of bacterial solution for 10<sup>9</sup> cells by using the formula V = 100 / (OD<sub>600</sub> × Dilution ratio).<br>
		+	(3) Take out a measured amount of 10<sup>9</sup> cells and centrifuge it at 8000 rpm for 3 min. Then pour out the supernatant.<br>
		+	(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.<br>
		+	(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℃.<br>
		+	(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.<br>
		+	(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room temperature.<br>
		+	【Day 3】Room temperature storage<br>
		+	【Day 4】Detect the survival rate<br>
		+	(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.<br>
		+	(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria solution on the LB plate.<br>
		+	(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after several gradient dilutions.<br>
		+	(4) Culture the bacteria overnight at 37℃.<br>
		+	【Day 5】Cell Count<br>
		+	(1) Take out the LB plate and take photos to record experimental results.<br>
		+	(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.
		+	</p>
		+
		+	<h4><b>Results:</b></h4>
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;First, by a reporter, sfGFP, we confirmed that the plasmid can normally expressed exogenous proteins in <i>E. coli</i> BL21 strain (Fig. 4).
		+
		+	[[File:T--QHFZ-China--pet28am sfGFP.png|400px|thumb|left|Figure 4. Via a reporter, sfGFP, the expression function of the modified pet28a vector was verified.]]
		+
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;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). </p>
		+
		+	[[File:T--QHFZ-China--TDP freeze-dry.png|400px|thumb|left|Figure 5. An independent repeat test of freeze-drying and recovery.]]
		+
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;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). </p>
		+
		+	[[File:T--QHFZ-China--OtsBA.png|400px|thumb|left|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.]]
		+
		+	<h4 style="clear:left;"><b>Summary: </b></h4>
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;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). </p>
		+
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here

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Latest revision as of 00:50, 27 October 2020

T7-Olac-RBS-OtsBA

    This biological part is the expression sequence of 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.

Contribution

Group: QHFZ-China iGEM 2020

Author: Yixian Yang

Design

    This part should be composed of a T7 promoter, RBS B0034, CDS of CAHS OtsBA BBa_K3457041, and a T7 terminator. However, we added another terminator B0015 before the T7 terminator. The reason is that if the T7 promoter is changed into other promoters, such as J23100, the corresponding RNA polymerase changed from T7 RNAP to endogenous RNAP of E. coli, which can be stopped by terminator B0015. In other words, the added terminator made the part easier to be modified. In the cartoon (Fig. 1), there are two points around the T7 promoter. They are two BsaI cutting site, which can be used to change the T7 promoter by Golden Gate technique.

    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.

    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 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).

<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).

    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).

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