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− | <h3 class="hh2" style="border:0;color:red" id="pos1">Part:BBa_K1668010</h3> | + | <h3 class="hh2" style="border:0;color:red" id="pos1">Part:BBa_K1668005</h3> |
− | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">plu1537-device</h4> | + | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">CDS <i>tcdA1</i></h4> |
| <p class="p1"> | | <p class="p1"> |
− | The part <i>plu1537</i> device is composed of arabinose inducible promoter pBad <a href="http://parts.igem.org/Part:">BBa_I0500</a>, toxin protein <i>plu1537</i> coding sequence <a href="http://parts.igem.org/Part:">BBa_K1668007</a> and composite part mCherry <a href="http://parts.igem.org/Part:">BBa_K1668011</a>. | + | The part CDS tcdA1 is the coding sequence of insecticidal protein tcdA1, which is used for termite control in our project. |
| + | |
| + | |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | We use the device to tandem express toxic protein Plu1537 and mCherry in<i> E.coli DH5α and BL 21 (DE3)</i>. The gene plu1537 is for termite control and mCherry is a reporter. | + | tcdA1 is one of the longest genes in bacteria. And the tcdA1 toxic protein is a 285kDa pore-forming protein, belonging to tc toxic family which is widely distributed among gram-positive and gram-positive bacteria. |
| </p> | | </p> |
| | | |
| | | |
| | | |
− | <div style="width:100%l;text-align:center;font-size:30px;"><a href="http://parts.igem.org/Part:BBa_K1668010">Sequence and Features</a></span> | + | <div style="width:100%l;text-align:center;font-size:30px;"><a href="http://parts.igem.org/Part:BBa_K1668005">Sequence and Features</a></span> |
| | | |
| | | |
− | | + | <h3 class="hh2">OVERVIEW</h4> |
− | | + | |
| | | |
− |
| |
− | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">Characterization</h4>
| |
− | <h5 class="hh2"> OVERVIEW </h5>
| |
| <p class="p1"> | | <p class="p1"> |
− | We build the device <i>plu1537</i> to express insecticidal toxic protein Plu1537 together with the reporter mCherry. The 14kDa insecticidal protein Plu1537 is used to kill termites in our project.
| + | tcdA1, one of the biggest proteins in bacteria (285kDa), is first found in Photorhabdus luminescens. |
| + | It forms channels and assists other toxins across the cell membrane(1). |
| + | It belongs to tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria. |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | The exact mechanism of Plu1537 remains unclear. In a 2009 research, scientists first expressed the plu1537 and purified it with GST tag. Consequently, Plu1537 showed its insecticidal activity against two moth species (Galleria& Spodoptera) larvae via hemocoel injection instead of oral feeding.
| + | We clone and standardize the gene into standard plasmid pSB1C3 and confirmed it by digesting and sequencing. |
| + | |
| </p> | | </p> |
| + | |
| + | <h3 class="hh2">Background</h4> |
| + | <div> |
| + | <div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/parts/3/35/ZJU-CHINA_tcdA1_structure.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Fig.1 The 3D structure of tcdA1. Copyright 2013, Nature Publishing Group. |
| + | </p> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | |
| <p class="p1"> | | <p class="p1"> |
− | We clone and standardize the gene into standard plasmid pSB1C3. After confirmation through enzyme digestion and whole sequencing, we transform the plasmid into <i>E.coli BL21 (DE3)</i> to achieve better expression level.
| + | tcdA1 is a pore-forming macro-protein, which can keep the ability to form a pore in a large pH range (from 4 to 11). To be noticed, at pH11, the pore-forming activity of tcdA1 is more than 100-fold greater than at pH6. As the midguts of most insects are alkaline, tc toxic proteins are effective by feeding on insects, including termites. |
| </p> | | </p> |
| + | |
| <p class="p1"> | | <p class="p1"> |
− | We observe that transformants have obviously turned red and we have identified the expected protein band in SDS-PAGE. According to <i>in vivo</i> test on termites, the toxic effect of Plu1537 is far beyond our expectation by oral feeding.
| + | In 2013, the structure of tcdA1 was revealed by researchers and reported in nature(1). As displayed in figure1a&b, the tcdA1 is composed of three parts: N-terminal a-helical domain(brown), the central b-sheet domain(green) and the C-terminal pore-forming domain(yellow). The protein has two states: pre-pore state and pore state. The pore-forming domain (figure 1c) sticks out to form pore, changing into pore state (figure 2). |
| </p> | | </p> |
− |
| + | <p class="p1"> |
− | <p class="p1">
| + | Moreover, the tcdA1 toxin helps other toxins to enter the cell membrane. Naturally in strain TT01, tcdA1 is expressed homologously with other toxins, for example, tcdB1 and tcc toxins. TcdA1 helps to transfer the latter into the cell to maximum the toxic effect(figure 3). |
− | In conclusion, we have successfully cloned and expressed the plu1537 in <i>DH5α BL 21(DE3)</i>. Toxic effects of oral feedings on termites are far better than that described in moth larvae research.
| + | |
| </p> | | </p> |
− | <h5 class="hh2"> BACKGROUND </h5> | + | |
| + | |
| + | <!-- 1668005 --> |
| + | <div> |
| + | <h3 class="hh2">Construction</h4> |
| + | <h4 class="hh4-left">PCR</h5> |
| <p class="p1"> | | <p class="p1"> |
− | In 2009 research<i> Expression and activity of a probable toxin from Photorhabdus Luminescens</i>, toxin protein pit, which is 94% homologous with plu1537, is expressed in <i>E.coli DH5α and BL21(DE3)</i>. Engineered strain <i>BL21</i> was both orally fed and injected in hemocoel to two kind of moth (Galleria mellonella & Spodoptera litura)(<i>1</i>). As a result, hemocoel injection is more effective than oral feeding. However, our experiment showed that oral feeding is also effectively. | + | The CDS<i>tcdA1</i> gene was amplified by PCR by three pieces to remove three enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. |
− | </p> | + | </p> |
| + | <p class="p1"> |
| + | We use primer tcdA1-left L and tcdA1-left R to amplify the left side of gene., tcdA1-middle L and tcdA1-middle R to amplify the middle part and tcdA1-right L and tcdA1-right R to amplify the right side. Primers are shown below. |
| + | </p> |
| + | <h4 class="hh4-left">Seamless assembly</h5> |
| <p class="p1"> | | <p class="p1"> |
− | Plu1537 shares 30% similarity with a 13.6 kDa insecticidal crystal protein cry34Ab1 in <i>Bacillus thuringiensis</i> in amino acid sequence (figure 1), which belongs to Bt toxin family.
| + | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, <i>tcdA1</i>-left, <i>tcdA1</i>-middle, <i>tcdA1</i>-right, and suffix sequence can be ligated seamlessly. |
| + | |
| + | |
| </p> | | </p> |
| + | <p class="p1 textcenter"> |
| + | tcdA1-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGAACTCGCCTGTAAAAGAGA </p> |
| + | <p class="p1 textcenter"> |
| + | tcdA1-left R (R, 5’-3’): AAATTCACAACAAGCGGATACATTACAC </p> |
| + | <p class="p1 textcenter"> |
| + | tcdA1-middle F(F, 5’-3’): TATCCGCTTGTTGTGAATTTAATCCGG </p> <p class="p1 textcenter"> |
| + | tcdA1-middle R(R, 5’-3’): TAACATTAGCGGAAAATTCCATCACATAACCTGTTGC </p> <p class="p1 textcenter"> |
| + | tcdA1-right F(F, 5’-3’): GGAATTTTCCGCTAATGTTATGAATACCGAAGC </p> <p class="p1 textcenter"> |
| + | tcdA1-right R(R, 5’-3’): CTGCAGCGGCCGCTACTAGTATTATTATTTAATGGTGTA </p> |
| + | |
| + | <h4 class="hh4-left">Transformation and confirmation</h5> |
| <p class="p1"> | | <p class="p1"> |
− | Bt family is a type of biological insecticidal toxin and may be the most famous protein family up to now. After years of study, Bt family is becoming bigger and bigger. Basically, most proteins in Bt family such as cry34Ab1(<i>2</i>) form pores in the membrane to kill the cell.
| + | After seamless assembly, standard plasmid pSB1C3 containing <i>tcdA1</i> gene was transformed into <i> E.coli DH5α</i>. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used tcdA1_M1(a primer on the right part of tcdA1)/ VR as the universal primers for the CDS<i>tcdA1</i> is too long to amplify all the parts sequence with VF2/VR .Primers are shown below. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing. |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | In 2014, the structure of cry34Ab1 was revealed and reported in PLOS ONE. As displayed in figure 1, the structure of cry34Ab1 is simpler, compared with other two toxins we used. Notably, cry34Ab1 (figure 2A) functions only with the assistant of cry35Ab1 (figure 2B)(<i>2</i>), while plu1537 performs its toxicity directly.
| + | tcdA1_M1(F, 5’-3’): CTGCCAATCTATGCCACACC </p> |
− | </p> | + | |
| + | <p class="p1"> |
| + | VR(F, 5’-3’): ATTACCGCCTTTGAGTGAGC </p> |
| + | |
| + | <h4 class="hh4-left">Plasmid map</h5> |
| <div class="col-md-12 textcenter"> | | <div class="col-md-12 textcenter"> |
− | <div class="col-md-6 img-100">
| + | <img src="https://static.igem.org/mediawiki/parts/7/78/ZJU-CHINA_TPCDS_A1.PNG" class="img-center" style="width:60%"></img> |
− | <img src="https://static.igem.org/mediawiki/parts/8/89/ZJU-CHINA_1537_3D_structure.png" class="img-center" style="height:883px"></img>
| + | <div class="cpleft"> |
− | <div class="cpleft">
| + | <p class="kuvateksti"> |
− | <p class="kuvateksti">
| + | Fig.2 The plasmid map of BBa_K1668005 CDS<i>tcdA1</i> |
− | Figure 1, the 3D structure of cry34Ab1. Copyright 2014, Worldwide Protein Data.
| + | </p> |
− | </p>
| + | |
| </div> | | </div> |
− | </div>
| + | </div> |
− | <div class="col-md-6 img-100">
| + | </div> |
− | <img src="https://static.igem.org/mediawiki/parts/d/d9/ZJU-CHINA_cry34Ab1.png" class="img-center"></img>
| + | |
− | <div class="cpright">
| + | <h3 class="hh2">RESULTS</h4> |
− | <p class="kuvateksti">
| + | <h4 class="hh4-left">DNA CONSTRUCTION</h5> |
− | Figure 2, comparison between cry34Ab1 and cry35Ab1(2). Copyright 2014, Public Library of Science.
| + | <div> |
| + | <div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/parts/5/57/ZJU-CHINA_CDSA1.png" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 4 PCR confirmation results of CDS tcdA1. We choose a 1k fragment in the middle of tcdA1 gene as template because the whole gene is much too long. |
| </p> | | </p> |
| </div> | | </div> |
− | </div>
| + | </div> |
− | </div> | + | </div> |
| + | |
| + | <p class="p1"> |
| + | The template we use is recombinant plasmids. |
| + | </p> |
| + | <p class="p1"> |
| + | It can be clearly seen the two recombinants shown in figure 4 is two positive cloning. |
| + | </p> |
| | | |
− |
| + | <h4 class="hh4-left">DNA SEQUNCING</h5> |
− | <div class="row"> | + | |
− | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">Results</h4>
| + | |
− | <div class="col-md-6 text-col-left">
| + | |
− |
| + | |
− | <h5 class="hh2"> PLASMID CONSTRUCTION</h5>
| + | |
− | <p class="p1">
| + | |
− | 5-μl samples of the single (L1) and double enzyme (L2) digestion products for plu1537-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (<a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>) for AGE parameters. We use PstI for single digestion, XbaI and PstI for double digestion, then products were determined by AGE analysis. The DNA size standards were the DL5,000 DNA Marker (M2; TaKaRa, Cat#3428A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.
| + | |
− | It can be clearly seen the plu1537 is constructed into the pSB1C3 backbone (figure 3).
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | <div class="col-md-6 img-100">
| + | |
− |
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/2/2f/ZJU-CHINA_1537_confirmation.png" class="img-center"></img>
| + | |
− | <div class="cpleft">
| + | |
− | <p class="kuvateksti">
| + | |
− | Figure 3 digestion confirmation of tcdA1-device in pSB1A2 backbone.
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− |
| + | |
− |
| + | |
− | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px"> Plasmid Sequencing </h4>
| + | |
| <p class="p1"> | | <p class="p1"> |
− | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 2.5k part shows 100% agreement with the desired sequence.
| + | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 7.5k part shows 100% agreement with the desired sequence. |
| + | </p> |
| + | <h3 class="hh2">Reference</h4> |
| + | <p class="p1"> |
| + | 1. C. Gatsogiannis et al., NATURE 495, 520 (2013-03-20, 2013). <br> |
| + | 2. D. Meusch et al., NATURE 508, 61 (2014). |
| </p> | | </p> |
− |
| |
− |
| |
− | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px"> Toxin Expression</h4>
| |
− | <h5 class="hh2"> BACTERIA CULTURE </h5>
| |
− | <div class="row">
| |
− | <p class="p1">
| |
− | The solid or liquid culture medium is LB culture with 34ug/ml chloromycetin and 80mM arabinose.
| |
− | </p>
| |
− | <p class="p1">
| |
− | Both the antibiotics and arabinose are added after the medium cools down to 60℃. 2% (w/v) of agar is added in solid medium.
| |
− | </p>
| |
− | <p class="p1">
| |
− | It can be clearly seen that the recombinant colonies turn red, indicating the expression of reporter mCherry (figure 4, 5). It is very likely that target gene is expressed.
| |
− | </p>
| |
− | <div class="col-md-6 img-100">
| |
− | <img src="https://static.igem.org/mediawiki/parts/4/45/ZJU-CHINA_plate_1537.png" class="img-center"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Figure 4 Expression of reporter mCherry in LB solid medium with arabinose and chloromycetin.
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | <div class="col-md-6 img-100">
| |
− |
| |
− |
| |
− | <img src="https://static.igem.org/mediawiki/parts/0/02/ZJU-CHINA_1537_EP_tube.png" class="img-center" style="height:496px"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Figure 5 Expression of reporter mCherry in LB liqiud medium with arabinose and chloromycetin.
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
− |
| |
− |
| |
− | <h5 class="hh2"> SDS-PAGE </h5>
| |
− | <div class="row">
| |
− | <div class="col-md-6 text-col-left">
| |
− | <p class="p1">
| |
− | 5uL sample is loaded in a 10% SDS-PAGE separation gel. The protein size standards was the 250kDa marker (M; Precision Plus Protein (Dual Color Standards),Cat#161-0374). See (<a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>) for more SDS-PAGE parameters.
| |
− | </p>
| |
− | <p class="p1">
| |
− | According to the result of SDS-PAGE (figure 6), target protein (14kDa) is highly expressed (line 4) compared with the negative control: the wildtype <i>BL21 (DE3)</i> strain (line 1).
| |
− | </p>
| |
− | </div>
| |
− | <div class="col-md-6 img-100">
| |
− |
| |
− | <img src="https://static.igem.org/mediawiki/parts/9/9d/ZJU-CHINA_Protein_expression.png" class="img-center"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Figure 6 SDS-PAGE results of four devices we constructed.
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
− | <h5 class="hh2"> TERMITES <i>in vivo</i> EXPERIMENTS </h5>
| |
− |
| |
− | <div class="row">
| |
− | <div class="col-md-12 textcenter">
| |
− |
| |
− | <img src="https://static.igem.org/mediawiki/2015/3/34/ZJU-china_plu1537_termite.png" class="img-center" style="width:60%"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− |
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
− |
| |
− |
| |
| | | |
| </article> | | </article> |
− |
| |
− |
| |
| | | |
| <article> | | <article> |
| | | |
− | <h3 class="hh2" style="border:0;color:red;padding-top:100px" id="pos2">Part: BBa_K1668009</h3> | + | <h3 class="hh2" style="border:0;color:red;padding-top:100px" id="pos2">BBa_K1668006</h3> |
− | <h4 class="hh4-left" style="padding-top:30px">plu0840-device</h4> | + | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">CDS plu0840</h4> |
| <p class="p1"> | | <p class="p1"> |
− | the part <i>plu0840</i> device consists of arabinose inducible promoter pBad <a href="http://parts.igem.org/Part:">BBa_I0500</a>, toxin protein <i>plu0840</i> coding sequence <a href="http://parts.igem.org/Part:">BBa_K1668006</a> and composite part mCherry <a href="http://parts.igem.org/Part:">BBa_K1668011</a>. Here we use the device to tandem express toxic protein Plu0840 and the reporter mCherry in<i> E.coli DH5α and BL 21(DE3)</i>.
| + | The part CDSplu0840 is coding sequence of toxin protein Plu0840, which is used for termite control in our project. |
| + | </p> |
| + | <p class="p1"> |
| + | Plu0840 is a 72kDa insecticidal toxic protein, which had weak oral toxicity against two kinds of moth according to a 2007 research and showed weak toxicity to termites by oral feeding. |
| </p> | | </p> |
| | | |
− | <div style="width:100%l;text-align:center;font-size:30px;"><a href="http://parts.igem.org/Part:BBa_K1668009">Sequence and Features</a></span>
| |
| | | |
− | | + | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">Characterization</h4> |
− | | + | <h3 class="hh2">OVERVIEW</h3> |
− | | + | |
− | <h4 class="hh4-left" style="padding-top:30px">Characterization</h4> | + | |
− | <h5 class="hh2"> OVERVIEW </h5> | + | |
| <p class="p1"> | | <p class="p1"> |
− | We construct the device <i>plu0840</i> to tandem express insecticidal toxic protein Plu0840 and reporter mCherry. The 72kDa insecticidal toxic protein Plu0840 is used to kill termites in our project. | + | Plu0840 is a insecticidal toxin found in Photorhabdus luminescens, a native toxin storehouse. In our project, it is used for termite control. |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | The exact mechanism of insecticidal toxin protein Plu0840 remains to be revealed. In a 2007 research, scientists first expressed a GST-plu1537 fusion protein in <i>E.coli BL21 (DE3)</i> with pGEX-4T-1 vector. The results showed that Plu0840 had weak oral toxicity against two kinds of moth (<i>S. litura and S. exigua</i>) | + | We clone and standardize the gene into standard plasmid pSB1C3, and confirmed the part by PCR and sequencing. Then we combine the CDS plu0840 with arabinose inducible promoter pBad in front reporter mCherry and double terminator behind into the device plu0840 to strongly express the toxin. |
| </p> | | </p> |
| + | <h3 class="hh2">BACKGROUND</h3> |
| <p class="p1"> | | <p class="p1"> |
− | We clone and standardize the gene into standard plasmid pSB1C3. After confirmation of digestion and sequencing, we transform the plasmid into <i>E.coli BL21 (DE3)</i> to achieve better expression level.
| + | In 2009 research Cloning and expression analysis of a predicted toxin gene from Photorhabdus sp. HB78, plu0840 fused with GST is expressed in Dh3α BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moth (S. litura and S.exigua)(1). |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | We observe that transformants have obviously turned red and figure out the expected protein band in SDS-PAGE. According to<i> in vivo </i>experiments on termites, the toxin effect of Plu0840 is comparatively weak than Plu1537, which is consistent with the research before.
| + | According to the result, on the one hand, oral feeding effectively inhibits the growth of larva while has only weak oral toxic effect. On the other hand, hemocoel injection showed negative results. |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | In conclusion, we have successfully cloned and expressed the Plu0840 toxic protein in <i>E.coli DH5α and BL 21(DE3)</i>. The Plu0840 is weak toxic to termites.
| + | The research mentions that Plu0840 (figure 1) shares 55% sequence identity with an enterotoxin Ast from aeromonas hydrophila. Aaeromonas hydrophila, which is connected with gastroenteritis, may lead to altered fluid secretion in mouse. According to a 2002 research, enterotoxin Ast makes weak contributions to fluid secretion compared with two other genes (2) However, judging that TT01 is nontoxic to animals at all, we think Plu0840 may share little similarity with Ast. |
| </p> | | </p> |
− | <h5 class="hh2"> BACKGROUND </h5> | + | <div> |
| + | <div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/parts/c/cd/ZJU-CHINA_0840_3D_structure.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 1 The 3D structure of Plu0840(4). Copyright 2014, Worldwide Protein Data. |
| + | </p> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | |
| + | |
| + | |
| + | <!-- 1668006 --> |
| + | <div> |
| + | <h3 class="hh2">Construction</h4> |
| + | <h4 class="hh4-left">PCR</h5> |
| <p class="p1"> | | <p class="p1"> |
− | In 2009 research<i> Cloning and expression analysis of a predicted toxin gene from Photorhabdus sp. HB78</i>, <i>plu0840</i> fused with GST is expressed in <i>E.coli DH5α and BL21 (DE3)</i>. Engineered strain <I>BL21</I> was both orally fed and injected in hemocoel to two kind of moth (<i>S. litura </i>and <i>S.exigua</i>)(<i>1</i>).
| + | The CDS<i>plu0840</i> gene was amplified by PCR by two pieces to remove one enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. <p class="p1"> |
− | </p>
| + | We use primer plu0840-left L and plu0840-left R to amplify the left side of gene. And we use plu0840-right L and plu0840-right R to amplify the right side. Primers are shown below. </p> |
| + | <h4 class="hh4-left">Seamless assembly</h5> |
| <p class="p1"> | | <p class="p1"> |
− | According to the result, on one hand, oral feeding effectively inhibits the growth of larva while has only weak oral toxic effect. On the other hand, hemocoel injection showed negative results.
| + | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, <i>plu0840</i>-left, <i>plu0840</i>-right, and suffix sequence can be ligated seamlessly. |
| + | |
| + | |
| + | |
| </p> | | </p> |
| + | <p class="p1 textcenter"> |
| + | plu0840-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGTTGTATAACACCCCAGT </p> |
| + | <p class="p1 textcenter"> |
| + | plu0840-left R (R, 5’-3’): CAACGAATTAAGAGGGAACAGCGGCC </p> |
| + | <p class="p1 textcenter"> |
| + | plu0840-right F(F, 5’-3’): TGTTCCCTCTTAATTCGTTGATGCTGCATGGC </p> <p class="p1 textcenter"> |
| + | plu0840-right R(R, 5’-3’): |
| + | ACTGCAGCGGCCGCTACTAGTATTATTATTTCGATGGGGTCAAAG |
| + | </p> |
| + | |
| + | <h4 class="hh4-left">Transformation and confirmation</h5> |
| <p class="p1"> | | <p class="p1"> |
− | The research mentions that Plu0840 (figure 1) shares 55% sequence identity with an enterotoxin Ast from <i>aeromonas hydrophila</i>. <i>Aaeromonas hydrophila</i>, which has something to do with gastroenteritis, may lead to altered fluid secretion in mouse. According to a 2002 research, enterotoxin Ast makes weak contributions to fluid secretion compared with two other genes (<i>2</i>). However, judging that <I>TT01</I> is nontoxic to animals at all, we think Plu0840 may share little similarity with Ast.
| + | After seamless assembly, standard plasmid pSB1C3 containing <i>plu0840</i> gene was transformed into <i> E.coli DH5α</i>. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing. </p> |
− | </p>
| + | <h4 class="hh4-left">Plasmid map</h5> |
− | <div class="row textcenter">
| + | <div class="col-md-12 textcenter"> |
− | <img src="https://static.igem.org/mediawiki/parts/c/cd/ZJU-CHINA_0840_3D_structure.jpg" class="img-center" style="width:60%"></img>
| + | <img src="https://static.igem.org/mediawiki/parts/f/f1/ZJU-CHINA_TPCDS_0840.PNG" class="img-center" style="width:60%"></img> |
− | <div class="cpleft">
| + | <div class="cpleft"> |
− | <p class="kuvateksti">
| + | <p class="kuvateksti"> |
− | Figure 1, the 3D structure of Plu0840. Copyright 2014, Worldwide Protein Data.
| + | Fig.2 The plasmid map of BBa_K1668006 CDS<i>plu0840</i> |
− | </p>
| + | </p> |
− | </div>
| + | |
− | </div>
| + | |
− | <div class="row"> | + | |
− | <h4 class="hh4-left" style="padding-top:30px">RESULTS</h4>
| + | |
− | <div class="col-md-6 text-col-left">
| + | |
− |
| + | |
− | <h5 class="hh2"> PLASMID CONSTRUCTION </h5>
| + | |
− | <p class="p1">
| + | |
− | 5-μl samples of the single (L1) and double enzyme (L2) digestion products for plu0840-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (<a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>) for AGE parameters. We use PstI for single digestion, XbaI and PstI for double digestion, then products were determined by AGE analysis. The DNA size standards were the DL5,000 DNA Marker (M2; TaKaRa, Cat#3428A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.
| + | |
− | </p>
| + | |
− | <p class="p1">
| + | |
− | It clearly indicates that the plu0840 is constructed into the pSB1C3 backbone (figure 2).
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | <div class="col-md-6 img-100">
| + | |
− |
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/a/af/ZJU-CHINA_0840_digest_confirmation.png" class="img-center"></img>
| + | |
− | <div class="cpleft">
| + | |
− | <p class="kuvateksti">
| + | |
− | Figure 2 digestion confirmation of device plu0840 in Psb1c3 backbone. | + | |
− | </p>
| + | |
| </div> | | </div> |
− | </div>
| + | </div> |
− | </div>
| + | </div> |
| | | |
| | | |
− | <h4 class="hh4-left" style="padding-top:30px"> PLASMID SEQUENCING </h4> | + | |
| + | |
| + | <h3 class="hh2">RESULTS</h3> |
| + | |
| + | <h3>PLASMID CONSTRUCTION</h3> |
| + | |
| <p class="p1"> | | <p class="p1"> |
− | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 3.9k part shows 100% agreement with the desired sequence.
| + | The template we use is recombinant plasmids. |
| + | </p> |
| + | <p class="p1"> |
| + | It can be clearly seen the recombinants shown in figure 2 is positive cloning. |
| </p> | | </p> |
− | <h4 class="hh4-left" style="padding-top:30px"> TOXIN EXPRESSION </h4> | + | <div> |
− | <h5 class="hh2"> BACTERIA CULTURE </h5>
| + | <div class="col-md-12 textcenter"> |
− | <div class="row">
| + | <img src="https://static.igem.org/mediawiki/parts/4/4a/ZJU-CHINA_CDS0840.png" class="img-center" style="width:60%"></img> |
− | <p class="p1">
| + | <div class="cpleft"> |
− | The solid or liquid culture medium is LB culture with 34ug/ml chloromycetin and 80mM arabinose. | + | <p class="kuvateksti"> |
− | </p>
| + | Figure 2 PCR confirmation results of CDS plu0840 with standard primers VF2 and VR. |
− | <p class="p1"> | + | </p> |
− | Both the antibiotics and arabinose are added after the culture cools down to 60℃. 2%(w/v) of agar is added in solid medium.
| + | </div> |
− | </p>
| + | </div> |
− | <p class="p1">
| + | </div> |
− | It can be clearly seen that the recombinant turned red, indicating the expression of reporter mCherry (figure 4, 5). As mCherry is located behind target gene and shares a promoter with target gene, the target gene may be expressed to a great extent.
| + | |
− | </p>
| + | |
| | | |
− | <div class="col-md-6 text-col-left">
| + | <h3>PLASMID SEQUNCING</h3> |
− |
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/d/db/ZJU-CHINA_0840_plate.jpg" class="img-center"></img>
| + | <p class="p1"> |
− | <div class="cpleft">
| + | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1.8k part shows 100% agreement with the desired sequence. |
− | <p class="kuvateksti">
| + | </p> |
− | Figure 3 expression of reporter mCherry in LB solid medium with arabinose and chloromycetin.
| + | <h3 class="hh2">REFERENCE</h3> |
− | </p>
| + | <p class="p1"> |
− | </div>
| + | 1. M. Li et al., MOL BIOL REP 36, 785 (2009). |
− | </div>
| + | </p> |
− | <div class="col-md-6 img-100">
| + | <p class="p1"> |
| + | 2. J. Sha, E. V. Kozlova, A. K. Chopra, INFECT IMMUN 70, 1924 (2002). |
| + | </p> |
| + | <p class="p1"> |
| + | 3. Plu0840 (uniprot):<a href="http://www.uniprot.org/uniprot/Q7N895" >http://www.uniprot.org/uniprot/Q7N895</a> . |
| + | </p> |
| + | <p class="p1"> |
| + | 4. 3D structure of Plu0840:<a href="http://www.proteinmodelportal.org/?pid=modelDetail&provider=MODBASE&template=1uasA&pmpuid=1000725544546&range_from=1&range_to=639&ref_ac=Q7N895&mapped_ac=Q7N895&zid=async" >Link Here</a> . |
| + | </p> |
| + | |
| + | </article> |
| | | |
− | <img src="https://static.igem.org/mediawiki/parts/6/6d/ZJU-CHINA_0840_EP_tube.jpg" class="img-center"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Figure 4 expression of reporter mCherry in LB liqiud medium with arabinose and chloromycetin.
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
− | <h5 class="hh2"> SDS-PAGE </h5>
| |
− | <div class="row">
| |
− | <div class="col-md-6 text-col-left">
| |
− | <p class="p1">
| |
− | 5uL sample is loaded in a 10% SDS-PAGE separation gel. The protein size standards was the 250kDa marker (M; Precision Plus Protein (Dual Color Standards),Cat#161-0374). See (<a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>) for more SDS-PAGE parameters.
| |
− | </p>
| |
− | <p class="p1">
| |
− | According to the result of SDS-PAGE (figure 5), target protein Plu0840 (72kDa) is strongly expressed (line 3) compared with the negative control: native BL21 (DE3) strain without engineering (line 1).
| |
− | </p>
| |
− | </div>
| |
− | <div class="col-md-6 img-100">
| |
− |
| |
− | <img src="https://static.igem.org/mediawiki/parts/9/9d/ZJU-CHINA_Protein_expression.png" class="img-center"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Figure 5 SDS-PAGE results of four devices we constructed.
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
− | <h5 class="hh2"> TERMITES <i>in vivo</i> EXPERIMENTS </h5>
| |
− | <div class="row">
| |
− | <div class="col-md-12 textcenter">
| |
− |
| |
− | <img src="https://static.igem.org/mediawiki/2015/b/be/ZJU-china_plu0840_termite.png" class="img-center" style="width:60%"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− |
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
− |
| |
− | </article>
| |
| | | |
| | | |
| <article> | | <article> |
| | | |
− | <h3 class="hh2" style="border:0;color:red;padding-top:100px" id="pos3">Part:BBa_K1668008</h3> | + | |
− | <h4 class="hh4-left" style="padding-top:30px">tcdA1 device</h4> | + | <h3 class="hh2" style="border:0;color:red;padding-top:100px" id="pos3">Part:BBa_K1668007</h3> |
| + | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">CDS <i>plu1537</i></h4> |
| <p class="p1"> | | <p class="p1"> |
− | the part tcdA1 device consists of arabinose inducible promoter pBad <a href="http://parts.igem.org/Part:">BBa_I0500</a>, toxin protein tcdA1 coding sequnce<a href="http://parts.igem.org/Part:">BBa_K1668005</a> and composite part mCherry <a href="http://parts.igem.org/Part:">BBa_K1668011</a>. | + | The part <i>CDSplu1537</i> is coding sequence of toxin protein Plu1537, which is used for termite control in our project. |
| + | |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | We use the device to tandem express toxic protein tcdA1 and mCherry. Toxic protein tcdA1 is a macro channel forming toxin used for termite control in our project and mCherry is a reporter. | + | Plu1537 is a 14kDa insecticidal toxic protein, which has strong toxicity against termites by oral feeding according to our termites experiment. It has 30% homology with a kind of Bt toxic protein, therefore it may play a similar role. |
| </p> | | </p> |
| | | |
| | | |
− | <!-- -->
| |
− | <div style="width:100%l;text-align:center;font-size:30px;"><a href="http://parts.igem.org/Part:BBa_K1668008">Sequence and Features</a></span>
| |
| | | |
| + | <div style="width:100%l;text-align:center;font-size:30px;"><a href="http://parts.igem.org/Part:BBa_K1668007">Sequence and Features</a></span> |
| + | |
| + | |
| + | |
| | | |
| | | |
| | | |
− | | + | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">Characterization</h4> |
− | <h4 class="hh4-left" style="padding-top:30px">Characterization</h4> | + | |
| <h5 class="hh2"> OVERVIEW </h5> | | <h5 class="hh2"> OVERVIEW </h5> |
| <p class="p1"> | | <p class="p1"> |
− | We construct the device tcdA1 to tandem express toxic protein tcdA1 and reporter mCherry. Toxic protein tcdA1 is used to kill termites in our project. | + | Plu1537 is a small insecticidal toxin found in Photorhabdus luminescens, a native toxin storehouse. In our project, it is used for termite control. |
| + | |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | tcdA1, one of the biggest proteins in bacteria (285kDa), is first found in Photorhabdus luminescens. It forms channels and assists other toxins to cross the cell membrane(1). It belongs to Tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria. | + | We clone and standardize the gene into standard plasmid pSB1C3, and confirmed the part by PCR and sequencing. Then we combine the CDS plu1537 with arabinose inducible promoter pBad in front reporter mCherry and double terminator behind into the device plu1537 to strongly express the toxin. Termites in vivo experiments results show that Plu1537 is strongly toxic to termites. |
| + | </p> |
| + | <h5 class="hh2"> BACKGROUND </h5> |
| + | <p class="p1"> |
| + | In 2009 research Expression and activity of a probable toxin from Photorhabdus Luminescens, toxin protein pit, which is 94% homologous with plu1537, is expressed in DH5α BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moths(Galleria mellonella & Spodoptera litura)(1). As a result, hemocoel injection is more effective than oral feeding. However, our experiment showed that oral feeding is also effective. </p> |
| + | <p class="p1"> |
| + | Plu1537 shares 30% amino acid sequence similarity with a 13.6 kDa insecticidal crystal protein cry34Ab1 in Bacillus thuringiensis (figure 1), which belongs to Bt toxin family. |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | We clone and standardize the gene into standard plasmid pSB1C3. After confirmation of digestion and sequencing, we transform the plasmid into <i>E.coli</i> BL21(DE3) to achieve better expression level. Despite we observe that transformants have obviously turned red, we didn’t figure out the expected protein band in SDS-PAGE. Judging that the protein is considerably huge in bacteria, more improvements are needed. | + | Bt family may be the most famous insecticidal toxin up to now. It’s one of biological toxins first used for pest control. After years of study, Bt family is becoming bigger and bigger. Basically, most proteins in Bt family form pores in cell membrane to kill a cell, including cry34Ab1(2). |
| </p> | | </p> |
− |
| + | <p class="p1"> |
− | | + | In 2014, the structure of cry34Ab1 was revealed and reported in PLOS ONE. As displayed in figure 1, the structure of cry34Ab1 is simpler, compared with other two toxins we used. However, cry34Ab1 (figure 2A) can only function with the assistant of cry35Ab1 (figure 2B)(2), which differentiate cry34 Ab1 from Plu1537, which is toxic without any other assistant. |
− | | + | </p> |
− | <div class="row"> | + | <div class="col-md-12 textcenter"> |
− | <h5 class="hh2"> BACKGROUND </h5>
| + | <div class="col-md-6 img-100"> |
− | <div class="col-md-6 text-col-left">
| + | <img src="https://static.igem.org/mediawiki/parts/7/76/Figure_2.png" class="img-center" style="height:883px"></img> |
− |
| + | |
− | <p class="p1">
| + | |
− | tcdA1 is a pore-forming macro-protein, which can keep the ability to form a pore in a large pH range (from 4 to 11). To be noticed, at pH11, the pore-forming activity of tcdA1 is more than 100-fold greater than at pH6. As the midguts of most insects are alkaline, Tc toxic proteins are effective by feeding on insects, including termites.
| + | |
− | </p> | + | |
− | <p class="p1">
| + | |
− | In 2013, the structure of tcdA1 was revealed by researchers and reported in nature(1). As displayed in figure1a&b, the tcdA1 is composed of three parts: N-terminal a-helical domain (brown), the central b-sheet domain (green) and the C-terminal pore-forming domain (yellow). The protein has two states: pre-pore state and pore state. The pore-forming domain (figure 1c) sticks out to form pore, changing into pore state (figure 2).
| + | |
− | </p>
| + | |
− | <p class="p1">
| + | |
− | Moreover, the tcdA1 toxin helps other toxins to enter the cell membrane. Naturally in strain TT01, tcdA1 is expressed homologously with other toxins, for example, tcdB1 and tcc toxins. TcdA1 helps to transfer the latter into the cell to maximum the toxic effect (figure 3).
| + | |
− | </p>
| + | |
− | </div>
| + | |
− |
| + | |
− | <div class="col-md-6 text-col-right"> | + | |
− |
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/3/35/ZJU-CHINA_tcdA1_structure.jpg" class="img-center"></img> | + | |
| <div class="cpleft"> | | <div class="cpleft"> |
| <p class="kuvateksti"> | | <p class="kuvateksti"> |
− | Figure 1, the 3D structure of tcdA1. Copyright 2013, Nature Publishing Group.
| + | Figure 1 Comparison between cry34Ab1 and cry35Ab1(2). Copyright 2014, Public Library of Science. |
− | </p>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | <div class="row">
| + | |
− | <div class="col-md-12 textcenter">
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/6/6d/ZJU-CHINA_A1_prepore_and_pore.png" class="img-center" style="width:60%"></img>
| + | |
− | <div class="cpleft">
| + | |
− | <p class="kuvateksti">
| + | |
− | Figure 2, comparison between pre-pore state and pore state of tcdA1 (2, 3). Copyright 2014, Nature Publishing Group
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | <div class="col-md-12 textcenter">
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/b/b6/ZJU-CHINA_tcdA1_transportation.jpg" class="img-center" style="width:60%"></img>
| + | |
− | <div class="cpleft">
| + | |
− | <p class="kuvateksti">
| + | |
− | Figure 3 the function of tcdA1 in toxin transportation (1). Copyright 2013, Nature Publishing Group.
| + | |
| </p> | | </p> |
| </div> | | </div> |
Line 437: |
Line 359: |
| | | |
| | | |
− | <div class="row">
| + | <!-- BBa_K1668007 --> |
− | <h4 class="hh4-left" style="padding-top:30px">RESULTS</h4>
| + | <div> |
− | <div class="col-md-6 text-col-left">
| + | <h3 class="hh2">Construction</h4> |
− |
| + | <h4 class="hh4-left">PCR</h5> |
− | <h5 class="hh2"> PLASMID CONSTRUCTION </h5>
| + | |
− | <p class="p1">
| + | |
− | 5μL samples of the double enzyme digestion products for tcdA1-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See<a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a> for AGE parameters. Sizes of the XbaI and PstI–cleaved assemblies were determined by AGE analysis. The DNA size standards were the DL5,000 DNA Marker (M2; TaKaRa, Cat#3428A) and 1kb DNA Ladder (Dye Plus)(M2; TaKaRa, Cat#3426A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.
| + | |
− | </p>
| + | |
− | <p class="p1">
| + | |
− | First we construct the tcdA1 device in pSB1A2. Our target fragments can be clearly seen in the right position (figure 4). As the fragment is a little big (7.2k), the efficiency is low when we change the backbone to pSB1C3 and the unwanted fragment is hard to explain (figure 5).
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | <div class="col-md-6 img-100">
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/3/3f/ZJU-CHINA_digestion_confirmation_A.png" class="img-center"></img>
| + | |
− | <div class="cpleft">
| + | |
− | <p class="kuvateksti">
| + | |
− | Figure 4 digestion confirmation of device tcdA1 in pSB1A2 backbone.
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | <img src="https://static.igem.org/mediawiki/parts/5/55/ZJU-CHINA_digestion_CONFIRMATION_C.png" class="img-center"></img>
| + | |
− | <div class="cpleft">
| + | |
− | <p class="kuvateksti">
| + | |
− | Figure 5 digestion confirmation of device tcdA1 in pSB1C3 backbone.
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | <h4 class="hh4-left" style="padding-top:30px"> PLASMID SEQUENCING </h4> | + | |
| <p class="p1"> | | <p class="p1"> |
− | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 9.7k part shows 100% agreement with the desired sequence.
| + | The CDS<i>plu0840</i> gene was amplified by PCR by two pieces to remove one enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. </p> |
| + | <p class="p1"> |
| + | We use primer plu0840-left L and plu0840-left R to amplify the left side of gene. And we use plu0840-right L and plu0840-right R to amplify the right side. Primers are shown below. </p> |
| + | <h4 class="hh4-left">Seamless assembly</h5> |
| + | <p class="p1"> |
| + | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, <i>plu0840</i>-left, <i>plu0840</i>-right, and suffix sequence can be ligated seamlessly. |
| + | |
| + | |
| + | |
| </p> | | </p> |
− | <h4 class="hh4-left" style="padding-top:30px"> TOXIN EXPRESSION </h4> | + | <p class="p1 textcenter"> |
| + | plu0840-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGTTGTATAACACCCCAGT </p> |
| + | <p class="p1 textcenter"> |
| + | plu0840-left R (R, 5’-3’): CAACGAATTAAGAGGGAACAGCGGCC </p> |
| + | <p class="p1 textcenter"> |
| + | plu0840-right F(F, 5’-3’): TGTTCCCTCTTAATTCGTTGATGCTGCATGGC</p> <p class="p1 textcenter"> |
| + | plu0840-right R(R, 5’-3’): |
| + | ACTGCAGCGGCCGCTACTAGTATTATTATTTCGATGGGGTCAAAG |
| + | </p> |
| + | |
| + | <h4 class="hh4-left">Transformation and confirmation</h5> |
| + | <p class="p1"> |
| + | After seamless assembly, standard plasmid pSB1C3 containing <i>plu0840</i> gene was transformed into <i> E.coli DH5α</i>. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing. </p> |
| + | <h4 class="hh4-left">Plasmid map</h5> |
| + | <div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/parts/f/f1/ZJU-CHINA_TPCDS_0840.PNG" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Fig.2 The plasmid map of BBa_K1668007 CDS<i>plu0840</i> |
| + | </p> |
| + | </div> |
| + | </div> |
| + | |
| | | |
− | <h5 class="hh2"> BACTERIA CULTURE </h5>
| |
| | | |
− | <div class="row">
| |
− | <p class="p1">
| |
− | The solid or liquid culture medium is LB culture with 34ug/ml chloromycetin and 80mM arabinose.
| |
− | </p>
| |
− | <p class="p1">
| |
− | Both the antibiotics and arabinose are added after the culture cools down to 60℃. 2%(w/v) of agar is added in solid medium.
| |
− | </p>
| |
− | <p class="p1">
| |
− | It can be clearly seen that the recombinant turned red, indicating the expression of reporter mCherry. As mCherry is located behind target gene and shares a promoter with target gene, the target gene may be expressed to a great extent.
| |
− | </p>
| |
− | <div class="col-md-6 text-col-left">
| |
− | <img src="https://static.igem.org/mediawiki/parts/7/77/ZJU-CHINA_tcdA1_plate.png" class="img-center"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Figure 6 expression of reporter mCherry in LB solid medium with arabinose and chloromycetin.
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | <div class="col-md-6 img-100">
| |
| | | |
− | <img src="https://static.igem.org/mediawiki/parts/0/04/ZJU-CHINA_tcdA1_EP_tube.png" class="img-center"></img>
| + | |
− | <div class="cpleft">
| + | </div> |
− | <p class="kuvateksti">
| + | |
− | Figure 7 expression of reporter mCherry in LB liqiud medium with arabinose and chloromycetin.
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | <h5 class="hh2"> SDS-PAGE </h5>
| + | |
| <div class="row"> | | <div class="row"> |
| + | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px">Results</h4> |
| <div class="col-md-6 text-col-left"> | | <div class="col-md-6 text-col-left"> |
| + | |
| + | <h5 class="hh2"> PLASMID CONSTRUCTION</h5> |
| <p class="p1"> | | <p class="p1"> |
− | 5uL sample is loaded in a 10% SDS-PAGE separation gel. The protein size standards was the 250kDa marker (M; Precision Plus Protein (Dual Color Standards),Cat#161-0374). See (<a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>) for more SDS-PAGE parameters.
| + | The template we use is recombinant plasmids. |
− | | + | |
− | </p>
| + | |
− | <p class="p1">
| + | |
− | According to the result of SDS-PAGE, target protein (285kDa) is not visible (line 2) compared with the negative control, native BL21 (DE3) strain without engineering (line 1). However, the recombinant tcdA1 strain turns red, indicating that it expressed mCherry, which can be confirmed in SDS-PAGE.
| + | |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | There are two possible explanations to the results. One is that the expression level of macro protein like tcdA1 is extremely low, which can not be unrecognized in SDS-PAGE. The other is that tcdA1 didn’t express out of unknown reason. | + | It can be clearly seen the recombinants shown in figure 3 is positive cloning. |
| + | |
| </p> | | </p> |
| </div> | | </div> |
| <div class="col-md-6 img-100"> | | <div class="col-md-6 img-100"> |
− | <img src="https://static.igem.org/mediawiki/parts/9/9d/ZJU-CHINA_Protein_expression.png" class="img-center"></img> | + | |
| + | <img src="https://static.igem.org/mediawiki/parts/c/c0/ZJU-CHINA_CDS1537.png" class="img-center"></img> |
| <div class="cpleft"> | | <div class="cpleft"> |
| <p class="kuvateksti"> | | <p class="kuvateksti"> |
− | Figure 8 SDS-PAGE results of four devices we constructed. | + | Figure 2 PCR confirmation results of CDS plu1537 with standard primers VF2 and VR. |
| </p> | | </p> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
− | <h5 class="hh2"> TERMITES <i>in vivo</i> EXPERIMENTS </h5>
| + | |
− |
| + | |
− | <div class="row">
| + | <h4 class="hh4-left" style="padding-top:30px" style="padding-top:30px"> DNA Sequencing </h4> |
− | <div class="col-md-12 textcenter">
| + | <p class="p1"> |
− |
| + | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1.8k part shows 100% agreement with the desired sequence. |
− | <img src="https://static.igem.org/mediawiki/2015/8/8e/ZJU-china_tcdA1_termite.png" class="img-center" style="width:60%"></img>
| + | |
− | <div class="cpleft">
| + | |
− | <p class="kuvateksti">
| + | |
− |
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | </div> | + | |
| | | |
− | </article> | + | </p> |
| + | |
| + | |
| + | <h3 class="hh2">Reference</h4> |
| + | <p class="p1"> |
| + | 1. M. Li et al., MOL BIOL REP 36, 785 (2009).<br> |
| + | 2. M. S. Kelker et al., PLOS ONE 9, (2014).<br> |
| + | 3. cry34Ab1(uniprot): http://www.uniprot.org/uniprot/Q939T0 <br> |
| + | 4. 3D structure of cry34Ab1: http://www.ebi.ac.uk/pdbe/entry/pdb/4JOX </p> |
| | | |
| | | |
− | <article>
| |
| | | |
− |
| + | </article> |
− | <h3 class="hh2" style="border:0;color:red" id = "pos4">BBa_K1668001: metK</h3>
| + | |
− | <h3 class="hh2">Overview</h4>
| + | |
− | <p class="p1">
| + | |
− | The part metK is the coding sequence of S-adenosylmethionine synthetase in Streptomyces avermitilis. It was found to stimulate the production of avermectins, one kind of pesticide.
| + | |
− | </p>
| + | |
− | <p class="p1">
| + | |
− | This gene sequence could not function in E.coli. If you would like to express metK in Streptomyces avermitilis, remember to add ermEp <a href="http://parts.igem.org/Part:BBa_K1668004">(BBa_K1668004)</a> as its promoter.
| + | |
− | </p>
| + | |
− | <h3 class="hh2">Background</h4>
| + | |
− | <p class="p1">
| + | |
− | metK is the gene encoding S-adenosylmethionine synthetase, which has been found in almost every organism. Its output catalyzes the formation of S-adenosylmethionine from methionine and ATP.
| + | |
− | </p>
| + | |
− | <h4 class="hh4-left">Function</h5>
| + | |
− | <p class="p1">
| + | |
− | In Streptomyces avermitilis, metK was found to stimulate the production of avermectins, a kind of pesticide. When wild-type S. avermitilis strain ATCC31267 was transformed with pYJ02 and pYJ03, two metK expression plasmids, avermectin production was increased about 2.0-fold and 5.5-fold compared with that in the control strains, respectively. (1)
| + | |
− | </p>
| + | |
− | <h4 class="hh4-left">Principle</h5>
| + | |
− | <p class="p1">
| + | |
− | As for the principle of improving the productivity, instead of changing cell growth or copy effect, metK stimulates the avermectin production by increasing the intracellular concentration of S-adenosylmethionine (SAM), an important intermediate product in avermectin production. However, there may be a maximum concentration of SAM for the production of avermectin in S. avermitilis, which means that SAM has no effect when its concentration achieve maximum.
| + | |
− | </p>
| + | |
− | <h4 class="hh4-left">Limitation</h5>
| + | |
− | <p class="p1">
| + | |
− | The results of experiments in research paper showed that different metK expression levels have different influence on avermectin production in various S. avermitilis strains. The gene expression levels of metK in two engineered strain, GB-165 and 76-05, were much higher than those in wild-type strain, whereas the avermectin productivity in these two strains have not been significantly improved(1). It is probably because the high expression level of metK in engineered strains limited the improvement of avermectin productivity by overexpression of metK.
| + | |
| | | |
| | | |
− | </p>
| |
− | <h4 class="hh4-left">Protein</h5>
| |
− | <p class="p1">
| |
− | The 3D structure of S-adenosylmethionine synthetase is as follows. This enzyme catalyzes the formation of S-adenosylmethionine from methionine and ATP and is involved in step1 of the subpathway that synthesizes S-adenosyl-L-methionine from L-methionine.
| |
− | </p>
| |
− | <div class="row"><div class="col-md-12 textcenter">
| |
− | <img src="https://static.igem.org/mediawiki/parts/f/f5/Zju-china-MetK-struc.jpeg"class="img-center" style="width:60%"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Fig.1 the 3D structure of S-adenosylmethionine synthetase (Uniprot #: Q3HW35)
| |
− | </p>
| |
− | </div>
| |
− | </div> </div>
| |
− | <h3 class="hh2">Construction</h4>
| |
− | <h4 class="hh4-left">PCR</h5>
| |
− | <p class="p1">
| |
− | The metK gene was amplified by PCR with genomic DNA extracted from S. avermitilis ATCC31267 strain as template. We commercially purchased this strain.
| |
− | </p>
| |
− | <p class="p1">
| |
− | By PCR with primers metK1 and metK2 shown below, we added the standard prefix and suffix at both ends of the metK sequence.
| |
− | </p>
| |
− | <h4 class="hh4-left">Seamless assembly</h5>
| |
− | <p class="p1">
| |
− | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our <a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>. By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly.
| |
− | </p>
| |
− | <p class="p1">
| |
− | metK1 (F, 5’-3’): GAATTCGCGGCCGCTTCTAGATGTTCGGCTACGC
| |
− | </p>
| |
− | <p class="p1">
| |
− | metK2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATTATTACAGCCCCACA
| |
− | </p>
| |
− | <h4 class="hh4-left">Transformation and confirmation</h5>
| |
− | <p class="p1">
| |
− | After seamless assembly, standard plasmid pSB1C3 containing metK gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2 and VR as the universal primers.
| |
− | </p>
| |
− | <p class="p1">
| |
− | The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.
| |
− | </p>
| |
− | <h4 class="hh4-left" style="font-size:30px">Plasmid map</h5>
| |
− | <div class="row"><div class="col-md-12 textcenter">
| |
− | <img src="https://static.igem.org/mediawiki/parts/5/56/Zju-china-BBa_K1668001_metK_Map.png"class="img-center" style="width:60%"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Fig.2 The plasmid map of BBa_K1668001 metK
| |
− | </p>
| |
− | </div>
| |
− | </div> </div>
| |
− | <h3 class="hh2">Results</h4>
| |
− | <h4 class="hh4-left">Gel electrophoretic analysis</h5>
| |
− | <p class="p1">
| |
− | In Fig.3 (A), it is indicated that metK (816bp) has been successfully amplified by PCR.
| |
− | </p>
| |
− | <p class="p1">
| |
− | In Fig.3 (B), positive clones determined by bacteria solution PCR are indicated.
| |
− | </p>
| |
− | <div class="row"><div class="col-md-12 textcenter">
| |
− | <img src="https://static.igem.org/mediawiki/parts/8/80/Zju-china-%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7_2015-09-14_%E4%B8%8B%E5%8D%8810.15.57.png"class="img-center" style="width:60%"></img>
| |
− | <div class="cpleft">
| |
− | <p class="kuvateksti">
| |
− | Fig.3 Gel electrophoretic analyses of PCR products (A) and selected examples of cloned products of seamless assembly reaction (B). (A) 5-μl samples of the PCR products for metK, (B) 5-μl samples of the bacteria solution PCR products were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. The DNA size standards was the DL1,000 DNA Marker (M1; Takara, Cat#3428A) and DL2,000 DNA Marker (M2; TaKaRa, Cat#3427A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager. PCR products, positive clones and negative clones are indicated.
| |
− | </p>
| |
− | </div>
| |
− | </div> </div>
| |
− | <h4 class="hh4-left">DNA sequencing</h5>
| |
− | <p class="p1">
| |
− | We have sequenced the parts with standard primers VF2 and VR. The sequence of the 816bp part shows 100% agreement with the desired sequence.
| |
− | </p>
| |
− | <h3 class="hh2">Reference</h4>
| |
− | <p class="p1">
| |
− | X. Zhao, Q. Wang, W. Guo, Y. Cai, C. Wang, S. Wang, S. Xiang, Y. Song, Overexpression of metK shows different effects on avermectin production in various Streptomyces avermitilis strains. World journal of microbiology & biotechnology 29, 1869-1875 (2013); published online EpubOct (10.1007/s11274-013-1350-0).
| |
− | </p>
| |
− |
| |
− | </article>
| |
| | | |
| | | |
Line 652: |
Line 457: |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp <a href="http://parts.igem.org/Part:BBa_K1668004">(BBa_K1668004)</a>as its promoter. | + | This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp<a href="http://parts.igem.org/Part:BBa_K1668004">(BBa_K1668004)</a>as its promoter. |
| </p> | | </p> |
| <h3 class="hh2">Background</h4> | | <h3 class="hh2">Background</h4> |
| <h4 class="hh4-left">Function</h5> | | <h4 class="hh4-left">Function</h5> |
| <p class="p1"> | | <p class="p1"> |
− | frr gene encodes the ribosome recycling factor (RRF), which is involved in the release of ribosomes from the translational post-termination complex for a new round of initiation. RRF may increase the efficiency of translation by recycling ribosomes from one round of translation to another. Avermectin yield was increased significantly by 3- to 3.7-fold in transformants 31267(pFRR-1139) and 31267(pFRRerm-1139), compared with that in the wild-type strains and both of the transformants contained multiple frr copies(1). The avermectin productivity of each culture was quantitatively measured by HPLC analysis. | + | frr gene encodes the ribosome recycling factor (RRF), which is involved in the release of ribosomes from the translational post-termination complex for a new round of initiation. RRF may increase the efficiency of translation by recycling ribosomes from one round of translation to another. Avermectin yield was increased significantly by 3- to 3.7-fold in transformants 31267(pFRR-1139) and 31267(pFRRerm-1139), compared with that in the wild-type strains and both of the transformants contained multiple frr copies.(1) The avermectin productivity of each culture was quantitatively measured by HPLC analysis. |
| </p> | | </p> |
| <h4 class="hh4-left">Principle</h5> | | <h4 class="hh4-left">Principle</h5> |
Line 665: |
Line 470: |
| <h4 class="hh4-left">Limitation</h5> | | <h4 class="hh4-left">Limitation</h5> |
| <p class="p1"> | | <p class="p1"> |
− | Compared with the wild-type strain, the effect of frr on avermectin production in engineered strains 76-02-e and GB-165 was less obvious, probably because most of the negative stimulatory factors are downregulated and most of the positive factors are upregulated, resulting in relatively limited potential for further improvement of avermectin yield(1). | + | Compared with the wild-type strain, the effect of frr on avermectin production in engineered strains 76-02-e and GB-165 was less obvious, probably because most of the negative stimulatory factors are downregulated and most of the positive factors are upregulated, resulting in relatively limited potential for further improvement of avermectin yield.(1) |
| </p> | | </p> |
| <h4 class="hh4-left">Protein</h5> | | <h4 class="hh4-left">Protein</h5> |
Line 686: |
Line 491: |
| <h4 class="hh4-left">Seamless assembly</h5> | | <h4 class="hh4-left">Seamless assembly</h5> |
| <p class="p1"> | | <p class="p1"> |
− | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our <a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>. By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly. | + | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol(此处应有超链接到protocol页). By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly. |
| </p> | | </p> |
− | <p class="p1"> | + | <p class="p1 textcenter"> |
| frr1 (F, 5’-3’): GAATTCGCGGCCGCTTCTAGATGCGCGGGTACGTC | | frr1 (F, 5’-3’): GAATTCGCGGCCGCTTCTAGATGCGCGGGTACGTC |
| </p> | | </p> |
− | <p class="p1"> | + | <p class="p1 textcenter"> |
| frr2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATTATTACATCAAGGTCGCC | | frr2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATTATTACATCAAGGTCGCC |
| </p> | | </p> |
Line 709: |
Line 514: |
| <h3 class="hh2">Results</h4> | | <h3 class="hh2">Results</h4> |
| <h4 class="hh4-left">Gel electrophoretic analysis</h5> | | <h4 class="hh4-left">Gel electrophoretic analysis</h5> |
− |
| + | <p class="p1"> |
| + | After overnight freeze-drying process, we got the final product CNCs in the end. (<b>Figure 3</b>) |
| + | </p> |
| <div class="col-md-12 textcenter"> | | <div class="col-md-12 textcenter"> |
− | | + | <img src="https://static.igem.org/mediawiki/2015/b/b9/ZJU-China_Result_CNCs3.jpg" class="img-center" style="width:60%"></img> |
| <div class="cpleft"> | | <div class="cpleft"> |
− | <p class="p1"> | + | <p class="kuvateksti"> |
| In Fig.3 (A), it is indicated that frr (384bp) has been successfully amplified by PCR. In Fig.3 (B), positive clones determined by bacteria solution PCR are indicated. | | In Fig.3 (A), it is indicated that frr (384bp) has been successfully amplified by PCR. In Fig.3 (B), positive clones determined by bacteria solution PCR are indicated. |
| </p> | | </p> |
Line 741: |
Line 548: |
| | | |
| | | |
− | <h3 class="hh2" style="border:0;color:red" id="pos6">BBa_K1668001: orfX</h3> | + | <h3 class="hh2" style="border:0;color:red" id="pos6">BBa_K1668003: orfX</h3> |
| <h3 class="hh2">Overview</h4> | | <h3 class="hh2">Overview</h4> |
| <p class="p1"> | | <p class="p1"> |
Line 747: |
Line 554: |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp <a href="http://parts.igem.org/Part:BBa_K1668004">(BBa_K1668004)</a> as its promoter. | + | This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp<a href="http://parts.igem.org/Part:BBa_K1668004">(BBa_K1668004)</a> as its promoter. |
| </p> | | </p> |
| <h3 class="hh2">Background</h4> | | <h3 class="hh2">Background</h4> |
| <h4 class="hh4-left">Function</h5> | | <h4 class="hh4-left">Function</h5> |
| <p class="p1"> | | <p class="p1"> |
− | The results of PCR analysis and the gene disruption experiments strongly suggest that either a considerably conserved sequence of orfX or a combination of orfX and other assisting genes exists in the high producers. And the orfX product appears to play an essential role in the production and regulation of avermectin in both the normal strain and the high producers. When wild-type S. avermitilis was transformed with a 8.0-kb DNA fragment containing the orfX gene, avermectin production increased approximately 3.5-fold(1). However, the nature of the stimulatory effect of orfX is still unclear. | + | The results of PCR analysis and the gene disruption experiments strongly suggest that either a considerably conserved sequence of orfX or a combination of orfX and other assisting genes exists in the high producers. And the orfX product appears to play an essential role in the production and regulation of avermectin in both the normal strain and the high producers. When wild-type S. avermitilis was transformed with a 8.0-kb DNA fragment containing the orfX gene, avermectin production increased approximately 3.5-fold.(1) However, the nature of the stimulatory effect of orfX is still unclear. |
| </p> | | </p> |
| <h4 class="hh4-left">Principle</h5> | | <h4 class="hh4-left">Principle</h5> |
| <p class="p1"> | | <p class="p1"> |
− | The orfX gene reveals a “copy number effect”. That is to say, multiple fragment copies can substantially increase avermectin production in S. avermitilis. Different from metK and frr gene, the DNA fragment containing orfX gene also increased avermectin bio-synthesis in various S. avermitilis strains, including the high-producing mutant strain ATCC 31780 and a semi-industrial strain L-9(1). | + | The orfX gene reveals a “copy number effect”. That is to say, multiple fragment copies can substantially increase avermectin production in S. avermitilis. Different from metK and frr gene, the DNA fragment containing orfX gene also increased avermectin bio-synthesis in various S. avermitilis strains, including the high-producing mutant strain ATCC 31780 and a semi-industrial strain L-9. (1) |
| </p> | | </p> |
| <h3 class="hh2">Construction</h4> | | <h3 class="hh2">Construction</h4> |
Line 765: |
Line 572: |
| <h4 class="hh4-left">Seamless assembly</h5> | | <h4 class="hh4-left">Seamless assembly</h5> |
| <p class="p1"> | | <p class="p1"> |
− | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our <a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>. By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly. | + | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our<a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>. By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly. |
| </p> | | </p> |
− | <p class="p1"> | + | <p class="p1 textcenter"> |
| orfX1 (F, 5’-3’): GAATTCGCGGCCGCTTCTAGATGGTGAGCGCCT | | orfX1 (F, 5’-3’): GAATTCGCGGCCGCTTCTAGATGGTGAGCGCCT |
| </p> | | </p> |
− | <p class="p1"> | + | <p class="p1 textcenter"> |
| orfX2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATTATTATCTGCGGTCC | | orfX2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATTATTATCTGCGGTCC |
| </p> | | </p> |
| <h4 class="hh4-left">Transformation and confirmation</h5> | | <h4 class="hh4-left">Transformation and confirmation</h5> |
| <p class="p1"> | | <p class="p1"> |
− | After seamless assembly, standard plasmid pSB1C3 containing orfX gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2 and VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing. | + | After seamless assembly, standard plasmid pSB1C3 containing orfX gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing. |
| </p> | | </p> |
| <h4 class="hh4-left">Plasmid map</h5> | | <h4 class="hh4-left">Plasmid map</h5> |
Line 821: |
Line 628: |
| | | |
| | | |
− | <h3 class="hh2" style="border:0;color:red" id="pos7">BBa_K1668001 ermEp</h3> | + | <h3 class="hh2" style="border:0;color:red" id="pos7">BBa_K1668004 ermEp</h3> |
| <h3 class="hh2">Overview</h4> | | <h3 class="hh2">Overview</h4> |
| <p class="p1"> | | <p class="p1"> |
Line 831: |
Line 638: |
| </p> | | </p> |
| <p class="p1"> | | <p class="p1"> |
− | The ermE promoter region contains two different promoters, ermEp1 and ermEp2. It was reported that a TGG deletion in the 35 region of the ermEp1 promoter resulted in a stronger variant called ermE* (ermEp2 and ermEp1 ΔTGG)(1). The promoter strength was indirectly assessed according to the enzymatic activity of the reporter protein GUS. The ermE and ermE* promoters were approximately 1.8 times stronger than the ermEp1 and ermEp1* promoters. However, no significant difference was detected between the strengths of the native ermE promoter and its variant ermE* or between the ermEp1 and the ermEp1* promoter(2). | + | The ermE promoter region contains two different promoters, ermEp1 and ermEp2. It was reported that a TGG deletion in the 35 region of the ermEp1 promoter resulted in a stronger variant called ermE* (ermEp2 and ermEp1 ΔTGG).(1) The promoter strength was indirectly assessed according to the enzymatic activity of the reporter protein GUS. The ermE and ermE* promoters were approximately 1.8 times stronger than the ermEp1 and ermEp1* promoters. However, no significant difference was detected between the strengths of the native ermE promoter and its variant ermE* or between the ermEp1 and the ermEp1* promoter.(2) |
| </p> | | </p> |
| <h3 class="hh2">Construction</h4> | | <h3 class="hh2">Construction</h4> |
Line 840: |
Line 647: |
| <h4 class="hh4-left">Seamless assembly</h5> | | <h4 class="hh4-left">Seamless assembly</h5> |
| <p class="p1"> | | <p class="p1"> |
− | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our <a href="https://2015.igem.org/Team:ZJU-China/Project/Protocol">Protocol</a>. By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly. | + | We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol(此处应有超链接到protocol页). By this way, prefix sequence, metK, and suffix sequence can be ligated seamlessly. |
| </p> | | </p> |
− | <p class="p1"> | + | <p class="p1 textcenter"> |
| ermEp1 (F, 5’-3’): ATTCGCGGCCGCTTCTAGAGGGCGGCTTGCGCC | | ermEp1 (F, 5’-3’): ATTCGCGGCCGCTTCTAGAGGGCGGCTTGCGCC |
| </p> | | </p> |
− | <p class="p1"> | + | <p class="p1 textcenter"> |
| ermEp2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATACCAACCGGCACGAT | | ermEp2 (R, 5’-3’): TGCAGCGGCCGCTACTAGTATACCAACCGGCACGAT |
| </p> | | </p> |
| <h4 class="hh4-left">Transformation and confirmation</h5> | | <h4 class="hh4-left">Transformation and confirmation</h5> |
| <p class="p1"> | | <p class="p1"> |
− | After seamless assembly, standard plasmid pSB1C3 containing ermEp gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2 and VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing. | + | After seamless assembly, standard plasmid pSB1C3 containing ermEp gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing. |
| </p> | | </p> |
| <h4 class="hh4-left">Plasmid map</h5> | | <h4 class="hh4-left">Plasmid map</h5> |
Line 858: |
Line 665: |
| <div class="cpleft"> | | <div class="cpleft"> |
| <p class="kuvateksti"> | | <p class="kuvateksti"> |
− | Fig.1 The plasmid map of BBa_K1668004 orfX | + | Fig.1 The plasmid map of BBa_K1668004 ermEp |
| </p> | | </p> |
| </div> | | </div> |
Line 904: |
Line 711: |
| | | |
| | | |
− |
| + | <p class="p1"> |
| + | Then, after centrifugation, we prepared the CNC Suspension. We used red laser pointer to irradiate DI water and the CNC Suspension, respectively, and only the CNCs forms the Tyndall effect, which proved the existence of CNCs conveniently. (<b>Figure 2</b>) |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/3/3f/ZJU-China_Result_CNCs2.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 2 CNC suspension |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <h3 class="hh2">Freeze-drying to get final product</h4> |
| + | <p class="p1"> |
| + | After overnight freeze-drying process, we got the final product CNCs in the end. (<b>Figure 3</b>) |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/b/b9/ZJU-China_Result_CNCs3.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 3 Solid CNCs |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <h3 class="hh2">Thermal Gravimetric Analyzer (TGA)</h4> |
| + | <p class="p1"> |
| + | TGA was carried out to observe the thermal characteristics of the CNCs (Figure 4). Evaporation of water led to the first stage of gradual weight loss. The onset temperature which CNCs began to degrade was around 223 ℃. The most obvious weight loss occurred at 393 ℃ while the literature value is 313 ℃[1], indicating the high thermal stability of CNCs we made. |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/f/f3/ZJU-China_Result_CNCs4.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 4 TGA analysis of CNCs |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <h3 class="hh2">TEM and SEM observation</h4> |
| + | <p class="p1"> |
| + | The pure CNC will crystallize in aqueous solutions and thus forming a square shape (<b>Figure 5</b>), which can be a standard to recognize whether bacteria are embedded in the CNC. |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/7/74/ZJU-China_Result_CNCs5.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 5 TEM images of CNCs |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <p class="p1"> |
| + | In the <b>Figure 6a</b>, <b>6b</b> taken under TEM, it’s obvious that the fibers of CNC are attached to the surface of E.coli, which reveals that the CNCs have successfully wrapped E.coli. Meanwhile the profile of CNCs has been displayed in <b>Figure 6c</b>, its sphere is extremely smooth while that of CNCs with E.coli is relatively rough. The red arrow of <b>Figure 6d</b> clearly indicates the location of E.coli. |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/d/da/ZJU-China_Result_CNCs6.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 6 TEM and SEM observation with E.coli |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <p class="p1"> In same process, we observed the embedding situation of Streptomycete as well. In the <b>Figure 7b</b>, different from the pure Streptomycete which has smooth fibers (<b>Figure 7a</b>), the embedding in CNCs results in Streptomycete’s surface having abundant granular substance (CNCs). On the other hand, the size of Streptomycete colony were extremely expanded after the embedding in CNCs (<b>Figure 7c, 7d</b>), which further revealed the success of embedding.</p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/e/ea/ZJU-China_Result_CNCs7.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 7 TEM and SEM observation with Streptomycete |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <p class="p1"> |
| + | The images of <b>Figure 8</b> show the growth of the CNC fibers. During the freeze-drying process, water infiltrated into the CNCs microspheres and formed multiple hydrogen bonds with CNCs, which caused the formation of mischcrystal under low temperature and had a structure of three-dimensional network. Water sublimated during freeze-drying so the porous CNCs skeleton was left. Therefore, we observed that the fibers formed by CNCs became more and more coiled while more and more slimy matters were adhered to the surface of the fibers with the increasing amount of bacteria. These proved the bacteria were embedded into the CNC fibers and had an indirect influence on the features of CNC fibers. |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/8/8a/ZJU-China_Result_CNCs8.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 8 CNC fibers with E.coli |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <h3 class="hh2">Dynamic Light Scattering (DLS)</h4> |
| + | <p class="p1"> |
| + | The Figure 9 reveals the embedding situation of E.coli with CNCs. E represents the pure E.coli. 4 h-CNC-E represent the microsphere of E.coli with CNCs. CNC is the microsphere with E.coli which has been stored in 4 ℃ for 20 days, and we guess the E.coli are dead and the CNC shell collapsed inward. |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/2/2f/ZJU-China_Result_CNCs9.jpg" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | Figure 9 Dynamic Light Scattering of E, CNC and 4 h-CNC-E |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <p class="p1"> |
| + | The <b>Figure 9</b> indicates that <b>4 h-CNC-E</b> occurs obviously self-assembly in general. The average particle sizes of each kind of compound are shown on the <b>Table 1</b>. |
| + | </p> |
| + | <div class="row"><div class="col-md-12 textcenter"> |
| + | <img src="https://static.igem.org/mediawiki/2015/4/40/ZJU-China_Result_CNCs10.png" class="img-center" style="width:60%"></img> |
| + | <div class="cpleft"> |
| + | <p class="kuvateksti"> |
| + | |
| + | </p> |
| + | </div> |
| + | </div> </div> |
| + | <p class="p1"> |
| + | Through simple subtraction, we can get the thickness of CNC on the surface of E.coli: |
| + | </p> |
| + | <p class="p1">Thickness = (1513.8 – 1317.1)/2 = 0.9835 nm</p> |
| + | <p class="p1">Reference</p> |
| + | <p class="p1">1 Zhou, J. et al. Synthesis of multifunctional cellulose nanocrystals for lectin recognition and bacterial imaging. Biomacromolecules 16, 1426-1432, doi:10.1021/acs.biomac.5b00227 (2015).</p> |
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| </article> | | </article> |
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| <ul id="nav2"> | | <ul id="nav2"> |
| <br> | | <br> |
− | <li><a href="#pos1" >BBa_K1668010 </a></li> <br> | + | <li><a href="#pos1" >BBa_K1668005 </a></li> <br> |
− | <li><a href="#pos2" >BBa_K1668009 </a></li> <br> | + | <li><a href="#pos2" >BBa_K1668006 </a></li> <br> |
− | <li><a href="#pos3" >BBa_K1668008 </a></li> <br> | + | <li><a href="#pos3" >BBa_K1668007 </a></li> <br> |
| <li><a href="#pos4" >BBa_K1668001:</br> metK </a></li> <br> | | <li><a href="#pos4" >BBa_K1668001:</br> metK </a></li> <br> |
| <li><a href="#pos5" >BBa_K1668002:</br> frr </a></li> <br> | | <li><a href="#pos5" >BBa_K1668002:</br> frr </a></li> <br> |
− | <li><a href="#pos6" >BBa_K1668001:</br> orfX </a></li> <br> | + | <li><a href="#pos6" >BBa_K1668003:</br> orfX </a></li> <br> |
− | <li><a href="#pos7" >BBa_K1668001:</br> ermEp</a></li> <br> | + | <li><a href="#pos7" >BBa_K1668004:</br> ermEp</a></li> <br> |
| | | |
| | | |