Difference between revisions of "Team:DUT China B/Experiments"

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               <h1  style="font-family: 'Times New Roman' !important; "><a name="Inspiration" >Inspiration </a><img src="https://static.igem.org/mediawiki/2019/9/98/T--DUT_China_B--INSPIRATION.svg" class="icon"> </h1>
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               <h1  style="font-family: 'Times New Roman' !important; "><a name="Inspiration" >1 Construction of expression vectors</a><img src="https://static.igem.org/mediawiki/2019/9/98/T--DUT_China_B--INSPIRATION.svg" class="icon"> </h1>
 
                  
 
                  
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<p style="font-family: 'Times New Roman' !important;  ">Have you ever seen the movie from MCU Antman? Imagine if the ant man's suit in the Marvel movie really exists in life，we can turn incredibly small, even to quantum scale, like the Antman who can get into Ironman’s suit and disable it in a minute, or sneak into a machine and fix it from the inside! We will have the opportunity to observe and even manipulate the world from an extremely microscopic perspective! Although the current technological development has not reached this level, it does not prevent us from exerting such bold imagination and trying to transform anything that might be used as a micro-robot. The cell is no doubt the finest complete living body and the most delicate control system we know. It is no exaggeration to compare it to a delicate life robot. But if we want natural cells to fully perform the functions we expect, the most viable way is to transform cells like machines. Cellular micro-robots have greatly attracted our attention, and we are excited to imagine such a steerable micro-robot and direct it for us. Synthetic biology is providing us with a way to build cell loops and transform cells according to our wishes, so this year, we can't wait to use synthetic biology to build such a controllable cell micro-nano robot.</p>
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<p style="font-family: 'Times New Roman' !important;  ">The plasmid of pOpt-clover-Paro (donated from professor Kong, DUT, China) was used as backbone for vector construction in our project. The green microalgae Chlamydomonas reinhardtii CC4533 from Chlamydomonas Resource Center (https://www.chlamycollection.org/) was used in our project. We coloned the gene of interest to this vector. The schematic representatives of vectors are shown as the following. In, first intron of the Chlamydomonas RbcS2 gene; RbcS2 T, Chlamydomonas RbcS2 terminator; Paro, Paromycin; HygB, Hygromycin B; VchR，Channelrhodopsins of Volvox；mCerulean3，a kind of cyan fluorescent proteins；Renilla luciferase，luciferin 2-monooxygenase from Renilla reniformis，N-hrluc-Pif3, fusion N-rluc with N-terminal phytochrome interacting factor 3; SP-PhyB-C-hrluc，fusion C-rluc with Phytochrome B.</p>
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<p>We also constructed the following constructs for expression in E.coli.N-nanoluc-Spytag-1, the combination of a new N terminal of Guassia luciferase and Spytag; SpyCatcher-C-nanoluc-1，the combination of new C terminal of Guassia luciferase and SpyCatcher；N-nanoluc-Spytag-2, see BBa_K1159201(链接); SpyCatcher-C-nanoluc-2, BBa_K1159200（链接）.</p>
 
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               <h1 style="font-family: 'Times New Roman' !important; ">Background <img src="https://static.igem.org/mediawiki/2019/b/b7/T--DUT_China_B--difficultities.svg" class="icon"> </h1>
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               <h1 style="font-family: 'Times New Roman' !important; ">2 Transformation and expression <img src="https://static.igem.org/mediawiki/2019/b/b7/T--DUT_China_B--difficultities.svg" class="icon"> </h1>
 
                  
 
                  
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<p style="font-family: 'Times New Roman' !important; ">When synthetic materials are difficult to meet the needs of control and loading, scientists have long thought of loading and modifying biological cells or molecules, using their own characteristics to operate on a small scale. Whether it is micro-nano manufacturing, precision medicine, single cell sorting or targeted drug loading, easy to manipulate micro-nano-scale robots have irreplaceable advantages. Depending on the invasiveness of the bacteria, the ability of the virus to transduce and self-replicate, the membrane encapsulation and drug loading capacity of the cells, different cells or biomolecules have been developed for micro-nano robots for specific application scenarios for targeted drug delivery. Or gene, cell therapy. The tiny size and control system of micro-nano robots make it a powerful tool for precision medical applications.</p>
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<p style="font-family: 'Times New Roman' !important; ">We introduced the genes of interest into C. reinhardtii CC4533 by electroporation, and obtained stable transformants through the screening of palonmycin or hygromycin B-resistant colonies. The target gene was introduced into E. coli DH5α (TaKaRa, Japan) by heat shock. For details, see Protocol 1 (链接).</p>
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              <p>Rigid micro-robots developed in the field of machine engineering have the best accuracy and control under programmable and automated operation, but are limited to the composition of mechanical control systems covering sensors, actuators and control circuits. The size of rigid robots is difficult to control in millimeters. Energy supply below the level and difficult to obtain wireless and reasonable output; poor biocompatibility of rigid mechanical materials also limits the development of mechanical micro-robots in the medical field. Compared with mechanical micro-nano robots, biological cells with micro-nano size and self-sufficient growth have a complete control system at the micro-nano scale, and the cell's own energy conversion system solves the micro-nano robot energy supply problem, which is also easier to carry out. Expression modification of drug proteins. </P>
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                <P> However, due to the uncertainty of the living body, the precise control of the cell micro-nano robot has become a major problem in the development of cell micro-nano robots. The control of cell mobility is one of the difficulties. One is limited to the weaker mobility of the cells themselves, and the other is limited to the sensing and motion control methods of the cells. At present, the commonly used methods are control of light, magnetism, material wrapping, etc. However, magnetic control requires more complicated external equipment and computer algorithms. The material wrapping needs to make more modifications to the cells, which may affect the activity of the cells, and may cause in vivo. Problems such as residual material modification, potential damage to the human body. Light control is a relatively convenient method of control, but still requires a more transparent application environment.</p>
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          <h1 style="font-family: 'JosefinSans-Light' !important; ">3 Measurement of the movement features of C. reinhardtii<img src="https://static.igem.org/mediawiki/2019/a/a2/T--DUT_China_B--wei.svg" class="icon"> </h1>
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            <p > A standard C. reinhardtii kinetics measurement protocol was established by
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              <h1 style="font-family: 'JosefinSans-Light' !important; ">Chlamydomonas reinhardtii<img src="https://static.igem.org/mediawiki/2019/a/a2/T--DUT_China_B--wei.svg" class="icon"> </h1>
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measuring the velocity of wild-type C. reinhardtii under blue light and determining the illuminance-velocity function.</p>
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<p >Chlamydomonas reinhardtii is a single-cell photoautotrophic eukaryote with the ability to accurately fold and assemble complex proteins. It can be used to express various complex proteins and high-value products. It is known as “green yeast”. It is said that the complete sequencing of the nuclear genome, chloroplast genome and mitochondrial genome and genetic transformation under three genomes are the most clear photosynthetic autotrophic eukaryotic substrates. Chlamydomonas cells have two flagellae, which are highly mobile and have a blue-light sensing system. They have the advantages of carrying protein-loading drugs and the potential for transformation using light control. They are characterized by the use of micro-nano robots. Good chassis creatures. Therefore, we hope to use Chlamydomonas as a chassis and modify it with its own strong mobility to solve the problem of mobility control of cellular nano-robots in applications.</p>
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                <img alt="" src="https://static.igem.org/mediawiki/2019/b/b5/T--DUT_China_B--move1.gif" style="display: inline-block;width:50%;" />               <p style="text-align: center;">Chlamydomonas reinhardtii with strong mobility</p>
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          <h1 style="font-family: 'JosefinSans-Light' !important; ">4 Characterization of the mutant channel rhodopsin VchR from the Volvox<img src="https://static.igem.org/mediawiki/2019/a/a2/T--DUT_China_B--wei.svg" class="icon"> </h1>
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                <p>The optical control system has the advantages of simple equipment, wireless control, good penetrability, etc., and Chlamydomonas itself has a blue light sensing system, so that the control of the movement of Chlamydomonas cells can be more fully utilized. Since cell micro-nano robots are mainly used in the medical field for targeted therapy, red light is more penetrating than other light tissues and is the most commonly used optical means in the medical field. Therefore, we hope to achieve red color in Chlamydomonas cells. The engineering of light control movement.</p>
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            <p > We characterized the effect of expression through measuring movement features of VchR-expressing C. reinhardtii. Measuring protocols see Protocol 5(链接).</p>
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                <p>To achieve the kinetic control of Chlamydomonas, how to transform its endogenous motion control and light perception system is the most effective means to achieve our transformation goals. However, because the movement of Chlamydomonas cells is controlled by two flagella, there are three different movement modes of swimming, fluctuation, and sliding under different conditions, and the movement mechanism is complicated. The specific molecular regulation network of two flagella in Chlamydomonas has not been obtained yet. Clear interpretation. Therefore, we cannot start from the molecular mechanism of the Chlamydomonasis movement. In the blue light sensing system of Chlamydomonas, we have learned that the eye spots of Chlamydomonas are used for blue light perception, and then the light signal is transmitted to the flagella to regulate the different movements of the two flagella under the action of the second messenger molecule. Therefore, we try to activate the Chlamydomonas light perception system from the light-gated ion channel at the eye spot to achieve the motion control of Chlamydomonas. But unfortunately, the mutants of the light-gated ion channels have limited redshift range, and we have not been able to find other substances that specifically activate or inhibit the rhodopsin of the Chlamydomonas channel, but only the general purpose of the cells. The second messenger molecule has a regulatory effect on it. We are unable to control the transformation of the universal messenger molecules in the cell, as this can distort the growth regulation of Chlamydomonas cells. So we have to give up on this idea.</P>
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            <p>After encountering a bottleneck in the molecular mechanism transformation, we tried to find a simpler way to control the algae.We have considered that since the channel of rhodopsin is excited by blue light, in the literature search, we have learned the research method of split protein and found the work of splitting luciferase for protein interaction. It is noted that the catalytic reaction of luciferase can produce blue light. We associate it with the possibility of combining red-controlled polymerized proteins with split luciferase. This enables the generation of blue light under red light control. We call this a molecular light converter. By expressing this molecular light converter in Chlamydomonas cells, we can achieve the excitation of endogenous blue light in Chlamydomonas cells, thus realizing the activation and motion control of Chlamydomonas light perception system. Our solution is thus generated.</P>
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        <td><img src="https://static.igem.org/mediawiki/2019/6/6c/T--DUT_China_B--molecular_light_converter_1.jpg" border=0></td>
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          <h1 style="font-family: 'JosefinSans-Light' !important; ">5 Effect of endogenous blue light on the trending movement of C. reinhardtii<img src="https://static.igem.org/mediawiki/2019/a/a2/T--DUT_China_B--wei.svg" class="icon"> </h1>
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            <p ><h3> 5.1 mCerulean3</h3>
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Observe the fluorescence of mCerulean3 expressing C. <i>reinhardtii</i> and explore the motion characteristics of mCerulean3 transformants.</p>
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          <p><h3> 5.2 Renilla luciferase  </h3>
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          <p style="text-align: center;">Molecular light converter</p>
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For the configuration, preservation and use of coelenterin solution, see protocol 4(链接).<br>
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Determination of optimal incubation time of Rluc. <br>
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Mix 2900 μL crude enzyme solution and 100 μL coelenterin working solution  and then measure the luminescence intensity at 480 nm every 5 min from 0up to 30 min.   <br>
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Determination of optimal substrate concentration of Rluc.<br>
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Mix appropriate amount of crude enzyme solution and the prepared coelenterin working solution, to make sure the working concentration coelenterin are 0, 5, 10, 15, 20 μM, respectively. After incubation for 10 min, measure the luminescence intensity at 480 nm.<br>
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Expression of Renilla luciferase in C. reinhardtii  <br>
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Transfer the pOpt-Rluc-paro plasmid to C. reinhardtii CC4533. 2700 μL of the culture solution was used, and after incubation with 300 μL of coelenterazine for 10 min, the intensity of the emitted light at 480 nm was measured.<br> </p>
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        <p><h3>5.3 Guassia luciferase</h3>
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st-1: the combination of new N terminal of Guassia luciferase and Spytag<br>
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st-2: the combination of N terminal of Guassia luciferase and Spytag  <br>
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sc-1: the combination of new C terminal of Guassia luciferase and SpyCatcher  <br>
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sc-2: the combination of C terminal of Guassia luciferase and SpyCatcher  <br>
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Directly homogenize st-1, st-2, sc-1, sc-2 engineered bacteria (IPTG induced) and collect supernatant crude enzyme solution by centrifugation. After determining the protein concentration, it is used for luminescence detection. Add 1350 μL each of these two interacting crude enzyme solutions in 3 mL system. Incubate for 10min and wait for the combination of Spytag and SpyCatcher, in order to restore the activity of Nanoluc. Add 300 μL coelenterazine solution and incubate it for 10 min to detect the emission light at 480 nm, so as to determine the activity of these two splitting sites.</p>  
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    <p><h3>5.4 PhyB/Pif3</h3>
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For the configuration, preservation and use of PCB solution, see protocol 3(链接).<br>
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Homogenize SP-PhyB-C-Rluc and N-Rluc-Pif3 engineered bacteria (IPTG induced) and collect supernatant crude enzyme solution by centrifugation. After determining the protein concentration, it is used for luminescence detection. After adding 1200 μL of these two proteins, they were incubated with 100 μL of PCB for 30 min, irradiated with red light for 5 min, and incubated with 300 μL of coelenterazine for 10 min, and the emission wavelength at 480 nm was measured.<br>
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Transfer the pOpt_SP-PhyB-C-Rluc_paro, pOpt_N-Rluc-Pif3_Hyg plasmids to Engineering C. reinhardtii CC4533 by electroporation. 2700 μL of the culture solution was taken, and after incubation with 300 μL of coelenterazine for 10 min, the intensity of the emitted light at 480 nm was measured. </p>        
 
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Revision as of 10:41, 21 October 2019

Document
parts

1 Construction of expression vectors

The plasmid of pOpt-clover-Paro (donated from professor Kong, DUT, China) was used as backbone for vector construction in our project. The green microalgae Chlamydomonas reinhardtii CC4533 from Chlamydomonas Resource Center (https://www.chlamycollection.org/) was used in our project. We coloned the gene of interest to this vector. The schematic representatives of vectors are shown as the following. In, first intron of the Chlamydomonas RbcS2 gene; RbcS2 T, Chlamydomonas RbcS2 terminator; Paro, Paromycin; HygB, Hygromycin B; VchR，Channelrhodopsins of Volvox；mCerulean3，a kind of cyan fluorescent proteins；Renilla luciferase，luciferin 2-monooxygenase from Renilla reniformis，N-hrluc-Pif3, fusion N-rluc with N-terminal phytochrome interacting factor 3; SP-PhyB-C-hrluc，fusion C-rluc with Phytochrome B.

We also constructed the following constructs for expression in E.coli.N-nanoluc-Spytag-1, the combination of a new N terminal of Guassia luciferase and Spytag; SpyCatcher-C-nanoluc-1，the combination of new C terminal of Guassia luciferase and SpyCatcher；N-nanoluc-Spytag-2, see BBa_K1159201(链接); SpyCatcher-C-nanoluc-2, BBa_K1159200（链接）.


nanorobot

2 Transformation and expression

We introduced the genes of interest into C. reinhardtii CC4533 by electroporation, and obtained stable transformants through the screening of palonmycin or hygromycin B-resistant colonies. The target gene was introduced into E. coli DH5α (TaKaRa, Japan) by heat shock. For details, see Protocol 1 (链接).

3 Measurement of the movement features of C. reinhardtii

A standard C. reinhardtii kinetics measurement protocol was established by measuring the velocity of wild-type C. reinhardtii under blue light and determining the illuminance-velocity function.

4 Characterization of the mutant channel rhodopsin VchR from the Volvox

We characterized the effect of expression through measuring movement features of VchR-expressing C. reinhardtii. Measuring protocols see Protocol 5(链接).

5 Effect of endogenous blue light on the trending movement of C. reinhardtii

5.1 mCerulean3

Observe the fluorescence of mCerulean3 expressing C. reinhardtii and explore the motion characteristics of mCerulean3 transformants.

5.2 Renilla luciferase

For the configuration, preservation and use of coelenterin solution, see protocol 4(链接).
Determination of optimal incubation time of Rluc.
Mix 2900 μL crude enzyme solution and 100 μL coelenterin working solution and then measure the luminescence intensity at 480 nm every 5 min from 0up to 30 min.
Determination of optimal substrate concentration of Rluc.
Mix appropriate amount of crude enzyme solution and the prepared coelenterin working solution, to make sure the working concentration coelenterin are 0, 5, 10, 15, 20 μM, respectively. After incubation for 10 min, measure the luminescence intensity at 480 nm.
Expression of Renilla luciferase in C. reinhardtii
Transfer the pOpt-Rluc-paro plasmid to C. reinhardtii CC4533. 2700 μL of the culture solution was used, and after incubation with 300 μL of coelenterazine for 10 min, the intensity of the emitted light at 480 nm was measured.

5.3 Guassia luciferase

st-1: the combination of new N terminal of Guassia luciferase and Spytag
st-2: the combination of N terminal of Guassia luciferase and Spytag
sc-1: the combination of new C terminal of Guassia luciferase and SpyCatcher
sc-2: the combination of C terminal of Guassia luciferase and SpyCatcher
Directly homogenize st-1, st-2, sc-1, sc-2 engineered bacteria (IPTG induced) and collect supernatant crude enzyme solution by centrifugation. After determining the protein concentration, it is used for luminescence detection. Add 1350 μL each of these two interacting crude enzyme solutions in 3 mL system. Incubate for 10min and wait for the combination of Spytag and SpyCatcher, in order to restore the activity of Nanoluc. Add 300 μL coelenterazine solution and incubate it for 10 min to detect the emission light at 480 nm, so as to determine the activity of these two splitting sites.

5.4 PhyB/Pif3

For the configuration, preservation and use of PCB solution, see protocol 3(链接).
Homogenize SP-PhyB-C-Rluc and N-Rluc-Pif3 engineered bacteria (IPTG induced) and collect supernatant crude enzyme solution by centrifugation. After determining the protein concentration, it is used for luminescence detection. After adding 1200 μL of these two proteins, they were incubated with 100 μL of PCB for 30 min, irradiated with red light for 5 min, and incubated with 300 μL of coelenterazine for 10 min, and the emission wavelength at 480 nm was measured.
Transfer the pOpt_SP-PhyB-C-Rluc_paro, pOpt_N-Rluc-Pif3_Hyg plasmids to Engineering C. reinhardtii CC4533 by electroporation. 2700 μL of the culture solution was taken, and after incubation with 300 μL of coelenterazine for 10 min, the intensity of the emitted light at 480 nm was measured.