Difference between revisions of "Part:BBa K3457040"

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	<partinfo>BBa_K3457040 short</partinfo>		<partinfo>BBa_K3457040 short</partinfo>
−	LEA	+	<p>&nbsp;&nbsp;&nbsp;&nbsp;This biological part is the CDS sequence of a Late Embryogenesis Abundant (LEA) protein, LEA3 protein, from soybean (Glycine max). It is a protein which help seeds resist dessication. </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--LEA basic design.png|400px|thumb|left|Figure 1. The Schematic cartoon of the DNA construct of BBa_K3457040.]]
		+
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;This part is the CDS of LEA3. We obtained the sequence from Group: iGEM10_Valencia. Then with the help of a company, we optimized the sequence, so that the part is suitable to work in <i>E. coli</i> and there is no biobrick restriction enzyme cutting site in it. Moreover, we added an 6x His tag at the N-terminal of it, so it can be easily detected by Western Blot and be purified by Ni-chelating affinity chromatography (Fig.1). This year, the part is used in the composite part [https://parts.igem.org/Part:BBa_K3457042 BBa_K3457042]</p>
		+
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;The part is similar to [https://parts.igem.org/Part:BBa_BBa_K401000 BBa_BBa_K401000].</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 LEA3 for comparison. </p>
		+
		+	<h4><b>Protocol: </b></h4>
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;To test the effect of LEA3, we modified a frequently and widely used vector, pet28a+ and put this part or TDP genes into it (Fig. 2). Then we transformed the plasmid into <i>E. coli</i> BL21 strain. </p>
		+
		+	[[File:T--QHFZ-China--pet28am.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 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.<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;">Then, by freeze-drying and recover experiment, we found that compared with the control group (sfGFP), the bacteria expressing LEA3 had a better survival rate (Fig. 5). We placed an enlarged picture as well (Fig. 7). </p>
		+
		+	[[File:T--QHFZ-China--TDP freeze-dry.png|400px|thumb|left|Figure 5. LEA3 protected bacteria during freeze-drying and subsequent dry storage.]]
		+	[[File:T--QHFZ-China--LEA.png|400px|thumb|left|Figure 6. An enlarged picture of fig. 5.]]
		+
		+	<h4 style="clear:left;"><b>Summary: </b></h4>
		+	<p style="clear:left;">&nbsp;&nbsp;&nbsp;&nbsp;LEA3 helps the bacteria survive freeze-drying and subsequent dry storage. To achieve that, you need only to transform the sequence into your bacteria. You can also use the part to express and purify LEA3 through Ni-chelating affinity chromatography.</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 00:39, 27 October 2020

LEA with a 6x His tag

    This biological part is the CDS sequence of a Late Embryogenesis Abundant (LEA) protein, LEA3 protein, from soybean (Glycine max). It is a protein which help seeds resist dessication.

Contribution

Group: QHFZ-China iGEM 2020

Author: Yixian Yang

Design

    This part is the CDS of LEA3. We obtained the sequence from Group: iGEM10_Valencia. Then with the help of a company, we optimized the sequence, so that the part is suitable to work in E. coli and there is no biobrick restriction enzyme cutting site in it. Moreover, we added an 6x His tag at the N-terminal of it, so it can be easily detected by Western Blot and be purified by Ni-chelating affinity chromatography (Fig.1). This year, the part is used in the composite part BBa_K3457042

    The part is similar to BBa_BBa_K401000.

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 LEA3 for comparison.

Protocol:

    To test the effect of LEA3, we modified a frequently and widely used vector, pet28a+ and put this part or TDP genes 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 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).

<p style="clear:left;">Then, by freeze-drying and recover experiment, we found that compared with the control group (sfGFP), the bacteria expressing LEA3 had a better survival rate (Fig. 5). We placed an enlarged picture as well (Fig. 7).

Summary:

    LEA3 helps the bacteria survive freeze-drying and subsequent dry storage. To achieve that, you need only to transform the sequence into your bacteria. You can also use the part to express and purify LEA3 through Ni-chelating affinity chromatography.

Sequence and Features