pGLO Plasmid Map and Resources

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Green Fluorescent Protein (GFP) isolated from jellyfish

Green fluorescent protein (GFP) is a protein that glows with a bright green fluorescence under ultraviolet light. First isolated from the marine jellyfish Aequorea victoria, the gene encoding GFP is used in cellular and molecular biology as a reporter to detect gene expression in transgenic organisms.

Bio-Rad Explorer pGLO Plasmid and GFP Kits use the pGLO plasmid, which contains the GFP gene, to enable hands-on learning about the central dogma, gene expression and regulation, bacterial transformation, protein separation, and the biomanufacturing process.

Bacterial Transformation

With pGLO bacterial transformation, students learn about genetic engineering as they transform a non-virulent laboratory strain of Escherichia coli (E. coli) with the pGLO plasmid. The procedure involves the CaCl2/heat shock method, which is a standard technique used in many research and biomanufacturing laboratories.

pGLO Plasmid

Bio-Rad’s pGLO plasmid contains DNA sequences that enable its replication and expression of the fluorescent trait (phenotype) in bacteria following transformation. The essential sequences include the following:

  • GFP — jellyfish gene that encodes green fluorescent protein (GFP)
  • ori — origin of pGLO plasmid DNA replication (essential for making more copies of the plasmid)
  • bla — gene that encodes β-lactamase, an enzyme that breaks down the antibiotic ampicillin; transformants expressing the bla gene can be selected by placing ampicillin in the growth medium
  • pBAD promoter — binds AraC-arabinose and promotes RNA polymerase binding and transcription of the GFP gene
  • araC — gene that encodes the regulatory protein that binds to the pBAD promoter; only when arabinose binds to the AraC protein is the production of GFP switched on
  • Multiple cloning site — a region containing restriction sites (NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion or deletion of a gene of interest

Bacteria transformed with the pGLO plasmid are selected by ampicillin resistance and when induced to express GFP, they glow fluorescent green under UV light!

Gene Regulation

Gene expression is carefully regulated to allow organisms to adapt to differing conditions and prevent wasteful production of proteins. Regulation often occurs at the level of transcription from DNA into RNA, specifically at the promoter, where RNA polymerase binds the DNA and begins transcription of the gene.

In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. These clusters of genes controlled by a single promoter are called operons. The bacterial genes encoding the enzymes needed to metabolize the simple sugar arabinose are a perfect example. Three genes that encode the digestive enzymes involved in breaking down arabinose (araB, araA, and araD) are clustered together in arabinose operon 3, and all depend on initiation of transcription from a single promoter, pBAD. Transcription requires the simultaneous presence of RNA polymerase, a DNA-binding protein called AraC, and arabinose.

  • When arabinose is absent, the AraC protein binds to the DNA at the binding site for RNA polymerase, preventing transcription of the digestive enzymes
  • When arabinose is present, it interacts with AraC, causing AraC to change shape, allowing RNA polymerase to bind the promoter; araB, araA, and araD are then expressed and can do their job to break down arabinose until the arabinose runs out

The pGLO plasmid contains both the promoter (pBAD) and araC gene, but araB, araA, and araD have been replaced by the single gene that codes for GFP, which serves as a reporter gene. In the presence of arabinose, the AraC protein promotes the binding of RNA polymerase to the promoter, which causes transcription of the GFP gene into messenger RNA (mRNA), followed by the translation of this mRNA into GFP. This process is called gene expression.

As they produce more and more protein, the cells expressing GFP fluoresce a brilliant green. In the absence of arabinose, however, AraC no longer facilitates the binding of RNA polymerase, and the GFP gene is not expressed, and bacterial colonies have a wild-type (natural) phenotype — white colonies with no fluorescence.

This is an excellent example of the central dogma of molecular biology in action: DNA > RNA > protein > trait.

Results of a pGLO bacterial transformation experiment

Results of a pGLO bacterial transformation experiment. Controls that were incubated with no plasmid (-pGLO) grow as a lawn in the absence of ampicillin (LB plate) and do not grow at all in the presence of ampicillin (LB/amp). Transformants grown on ampicillin (LB/amp) grow as colonies but do not show GFP fluorescence; those grown in the presence of both amp and ara (LB/amp/ara) do glow green under UV light.

Green Fluorescent Protein

Green Fluorescent Protein (GFP) barrel structure

GFP has a barrel structure surrounding a central alpha helix that contains the fluorophore. It can be used as an example for discussions of protein secondary structure, parallel and anti-parallel beta sheets, and the use of genes and proteins in biotechnology. For more information, visit our partner 3-D Molecular Designs.


GFP Purification with Chromatography and Electrophoresis

GFP Purification — Electrophoresis and Chromatography (PPT 9.63 MB)

The GFP expressed from the pGLO plasmid illustrates the central doctrine of biology, from the transformation of DNA to the expression of a protein to the visualization of a trait. The bacterial proteome contains thousands of proteins, but only the cloned GFP glows!

In its native environment, GFP fluoresces in the deep sea jellyfish, Aequorea victoria. Incredibly, GFP retains its fluorescent properties when cloned and expressed in E. coli and even when isolated from E. coli and separated on polyacrylamide gels or by chromatography. These amazing properties of GFP allow students to visualize the phenotypic properties of a protein and identify the single protein “band” responsible for the trait. These extensions link two of the most commonly used techniques in biotechnology labs: transformation and protein purification.

Purification of a protein depends on a its chemical or physical properties, such as molecular weight, electrical charge, or solubility. GFP can be separated from others by its size using electrophoresis, and it is extremely hydrophobic, which enables its purification using hydrophobic interaction chromatography (HIC). When placed in a buffer containing a high concentration of salt, the HIC matrix selectively binds hydrophobic GFP molecules while allowing the bacterial proteins to pass right through the column. Then, simply lowering the salt concentration of the buffer causes GFP to elute from the column in a purer form.

Students can explore these separation techniques by growing transformed bacteria in liquid culture to grow overnight, then lysing the cells to release their contents. The unique fluorescent property of GFP allows real-time monitoring of extraction and purification, modeling key processes used in biotechnology to produce and purify designer proteins with commercial or research value.

Other Resources

 

Case Studies

Student-facing extensions that are also useful for AP exam prep.

pGLO Bacterial Transformation and GFP Kits

pGLO Bacterial Transformation Kit

pGLO Lab Kits utilize Bio-Rad’s pGLO plasmid, which encodes a green fluorescent protein (GFP), to enable instructors to give students a hands-on introduction to transformation, cloning, protein chromatography, and electrophoresis techniques.

pGLO Bacterial Transformation Kit

Use bacterial transformation with an inducible promoter to make glowing
E. coli.

pGLO Transformation and Inquiry Kit for AP Biology

Investigate the functional elements of pGLO bacterial transformation, including heat shock, antibiotic selection, promoters, and satellite colony formation.

pGLO SDS-PAGE Extension

Use protein electrophoresis to view the expression of proteins in your pGLO bacteria.

Green Fluorescent Protein Chromatography Kit

Use chromatography to purify glowing green fluorescent protein from your pGLO bacteria.

Secrets of the Rainforest Kit

Simulate the drug discovery process in your classroom with a glowing protein.

NISQOC15 [x-forwarded-proto] = [https] [amp-cache-transform] = [google;v="1..3"] [x-forwarded-port] = [443] [x-forwarded-for] = [66.249.66.25, 10.232.3.244] [accept] = [text/html,application/xhtml+xml,application/signed-exchange;v=b3,application/xml;q=0.9,*/*;q=0.8] [seourl] = [/en-us/applications-technologies/pglo-plas-map-resources] [x-amzn-trace-id] = [Root=1-5edb43b9-ee7a37b3fe73f0e7fe808a80] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=NISQOC15] [host] = [10.232.0.21:1776] [x-request-uri] = [/en-us/applications-technologies/pglo-plas-map-resources] [from] = [googlebot(at)googlebot.com] [connection] = [Keep-Alive] [accept-encoding] = [gzip,deflate,br] [user-agent] = [Mozilla/5.0 (Linux; Android 6.0.1; Nexus 5X Build/MMB29P) AppleWebKit/537.36 (KHTML, like Gecko) Chrome/80.0.3987.92 Mobile Safari/537.36 (compatible; Googlebot/2.1; +http://www.google.com/bot.html)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/pglo-plas-map-resources LSE NISQOC15 pGLO Plasmid Map Sequence and Overview of pGLO Gene Regulation pGLO Plasmid Map and Resources /webroot/web/html/lse/solutions/applications/preparing_for_class <script type="text/javascript">// <![CDATA[ if ($.browser.msie && $.browser.version < 8) {$("div.methodboxmiddle ul.rightarrowsearch1").css( {"margin-left":"-5px"} );} $('head').append('<meta name="DCSext.at_banner" content="qPCR/Real-Time PCR" /><meta name="DCSext.at_banner_e" content="v" />'); $(document).ready(function () { $("#rightcol").append('<div id="relatedProducts"> </div>'); $('#relatedProducts').load('/webroot/web/html/lsr/solutions/technologies/pcr/related-products-pGLO-Plasmid-Map-and-Resources.html'); }); // ]]></script> <!--<div class="corner-box corner-box-promo corner-box-promo-edu linkpointer"><img src="/webroot/web/images/lse/support/pGLO-callout_img_160x1601.jpg" border="0" alt="DNA gels" caption="false" width="64" height="64" /> <p><strong><b>Looking for the pGLO Bacterial Transformation Kit?</b></strong></p> <div class="bttn-block"><a class="bttn-block no-target-style" href="/en-us/category/pglo-plasmid-gfp-kits?ID=f75948d2-dc20-4a32-b4e5-b7e0fe4c21ed&amp;WT.mc_id=191014027131" target="_blank" rel="noopener noreferrer"><span class="bttn-orange">See Kits</span> </a> <a class="bttn-orange no-target-style" href="/en-us/product/pglo-bacterial-transformation-kit?ID=619b8f74-9d3f-4c2f-a795-8a27e67598b7&amp;WT.mc_id=191014027131" target="_blank" rel="noopener noreferrer"><span class="bttn-orange">Buy Refills</span> </a></div> </div>--> <p>Green fluorescent protein (GFP) is a protein that glows with a bright green fluorescence when exposed to ultraviolet light. The gene sequence coding for GFP was first isolated from the marine jellyfish <em>Aequorea victoria</em> and has since been widely used in cellular and molecular biology as a reporter to detect gene expression in transgenic organisms. The pGLO plasmid contains the GFP gene and other functional DNA sequences that enable its use for bacterial transformation and expression.</p> pGLO Gene Regulation: One Gene, One Protein? <p>Our bodies contain thousands of different proteins, which perform many different jobs. Digestive enzymes, some of the antibodies protecting us from disease, and the hormone signals that run through our bodies are all proteins. The information for assembling a protein is carried in our DNA. The section of DNA that contains the code for making a protein is called a gene. There may be up to 100,000 genes in the human genome. Each gene codes for a unique protein: one gene, one protein. The gene that codes for a digestive enzyme in your mouth is different from one that codes for an antibody or a pigment that colors your eyes.</p> <p>Organisms regulate the expression of their genes and, ultimately, the amounts and kinds of proteins present within their cells, for a myriad of reasons, including development, cellular specialization, and adaptation to the environment. Gene regulation not only allows adaptation to differing conditions but also prevents wasteful overproduction of unneeded proteins, which would put the organism at a competitive disadvantage. The genes involved in the transport and breakdown (catabolism) of food are good examples of highly regulated genes. For example, the sugar arabinose is both a source of energy and a source of carbon.&nbsp;<em>E. coli</em>&nbsp;bacteria produce three enzymes (proteins) needed to digest arabinose as a food source. The genes that code for these enzymes are not expressed when arabinose is absent, but they are expressed when arabinose is present in their environment. How is this so?</p> <p>Regulation of protein expression often occurs at the level of transcription from DNA into RNA. This regulation takes place at a very specific location on the DNA template, called a promoter, where RNA polymerase binds the DNA and begins transcription of the gene. In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. These clusters of genes controlled by a single promoter are called operons.</p> <p>The three genes that code for the three digestive enzymes involved in breaking down arabinose (<em>araB</em>, <em>araA</em>, and <em>araD</em>) are clustered together in what is known as arabinose operon 3. These three proteins depend on initiation of transcription from a single promoter, pBAD. Transcription of these three genes requires the simultaneous presence of the DNA template (promoter, gene, and operon), RNA polymerase, a DNA-binding protein called AraC, and arabinose. The AraC protein binds to the DNA at the binding site for RNA polymerase (the beginning of the arabinose operon).</p> <p>When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with AraC, which is bound to the DNA. The interaction causes AraC to change shape, which in turn helps RNA polymerase bind the promoter, and the three genes <em>araB</em>, <em>araA</em>, and <em>araD</em> are transcribed. Three enzymes are produced; they do their job to break down arabinose, and eventually the arabinose runs out.</p> <p>The DNA sequence of the pGLO plasmid has been engineered to incorporate aspects of the arabinose operon. Both the promoter (pBAD) and the <em>araC</em> gene are present. However, the genes that code for arabinose catabolism (<em>araB</em>, <em>araA</em>, and <em>araD</em>) have been replaced by the single gene that codes for the green fluorescent protein (GFP), which serves as a reporter gene. In the presence of arabinose, the AraC protein promotes the binding of RNA polymerase to the promoter, which causes the transciption of the <em>GFP</em> gene into messenger RNA (mRNA), followed by the translation of this mRNA into GFP; this process is called gene expression.</p> <p>As they produce more and more protein, the cells expressing GFP fluoresce a brilliant green. In the absence of arabinose, AraC no longer facilitates the binding of RNA polymerase, and the <em>GFP</em> gene is not expressed. When the GFP protein is not made, bacterial colonies will appear to have a wild-type (natural) phenotype &mdash; of white colonies with no fluorescence. This is an excellent example of the central dogma of molecular biology in action: DNA &gt; RNA &gt; protein &gt; trait.</p> <div class="top"><a href="http://usherlxp03.hdc.bio-rad.com/iw-cc/command/#helptop">Back to Top</a></div> pGLO Plasmid Map <a name="plasmidmap"></a> <p>Our unique pGLO plasmid map is available for educational use only.</p> <p><img src="http://www.bio-rad.com/webroot/web/images/lse/products/programs/pglomap2.jpg" border="0" alt="" /></p> <p><a href="/webroot/web/pdf/lse/misc/pGLO_plasmid_sequence.txt">Download pGLO DNA Sequence</a>&nbsp;(Plain text file)</p> <p>pGLO contains several DNA sequences that enable replication of the plasmid DNA and expression of the fluorescent trait (phenotype) in bacteria following transformation. The essential sequences include the following:</p> <p><strong><em>GFP</em>&nbsp;</strong>&mdash; the jellyfish gene that codes for the production of green fluorescent protein</p> <p><em><strong>bla</strong></em>&nbsp;&mdash; a gene that encodes the enzyme beta-lactamase, which breaks down the antibiotic ampicillin. Bacteria containing the <em>bla</em> gene can be selected by placing ampicillin in the growth medium.</p> <p><strong>ori</strong>&nbsp;&mdash; the origin of pGLO plasmid DNA replication</p> <p><em><strong>araC</strong></em>&nbsp;&mdash; a gene that encodes the regulatory protein that binds to the pBAD promoter. Only when arabinose binds to the AraC protein is the production of GFP switched on.</p> <p><strong>pBAD promoter</strong>&nbsp;&mdash; a specific DNA sequence upstream from the GFP gene, which binds araC-arabinose and promotes RNA polymerase binding and transcription of the GFP gene.</p> <p><strong>Multiple cloning site </strong>&mdash; a region containing restriction sites (NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion or deletion of the gene of interest</p> <div class="top"><a href="#helptop">Back to Top</a></div> More about GFP <div style="float: left; margin-right: 20px;"><img src="http://www.bio-rad.com/webroot/web/images/lse/products/programs/bioed_gfp.jpg" alt="" /></div> <p>For additional information and imagery that involve the unique properties of GFP, we recommend that you visit the website developed and maintained by the Center for BioMolecular Modeling at the Milwaukee School of Engineering:&nbsp;<a class="no-target-style" href="http://cbm.msoe.edu/teachRes/jmol/" target="_blank" rel="noopener noreferrer">GFP 3-D images.</a></p> <div class="top"><a href="#helptop">Back to Top</a></div> Life Science Education/Products/pGLO/GFP Kits/pGLO Bacterial Transformation Kit ->MTS::NISQOC15##Life Science Education/Products/pGLO/GFP Kits/Green Fluorescent Protein Chromatography Kit ->MT::7f20553c-73f8-498a-b0b1-75d985308bf8##Life Science Education/Products/pGLO/GFP Kits/Secrets of the Rainforest Kit ->MT::6b225c3a-5f77-4680-8b34-1361fecf8f4e##Life Science Education/Products/pGLO/GFP Kits/pGLO SDS-PAGE Extension ->MT::a41608e9-b348-43e0-98bb-d0ae12664e06## Karen Moss pGLO Plasmid Map Green fluorescent protein (GFP) is a protein that glows with a bright green fluorescence under ultraviolet light. Bio-Rad Explorer pGLO Plasmid and GFP Kits use the pGLO plasmid, which contains the GFP gene, to enable hands-on learning about the central dogma, gene expression and regulation, bacterial transformation, protein separation, and the biomanufacturing process."> <meta property="og:title" content="pGLO Plasmid Map and Resources" /> <meta property="og:description" content="Explore Bio-Rad’s array of free teaching resources that range from online videos to editable presentations and student activities that can stand alone or enrich your lessons." /> <meta property="og:url" content="/en-us/applications-technologies/pglo-plasmid-map-resources?ID=NISQOC15" /> <meta property="og:site_name" content="Bio-Rad Laboratories" /> <meta property="og:image" content="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/GFP-pglo-jellyfish.jpg" /> <meta property="pinterest:richpins" content="enabled 01/26/15 12:24 PM 01/27/25 12:26 AM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA,VN en LSE /LSE/Applications/Preparing_for_Class N 0 <div class="pagenav"><img src="/webroot/web/images/opt/prodnav-arrow-top.png" alt="" /> <table border="0" role="presentation"> <tbody> <tr> <td class="pagenavhead"> <h3>On This Page</h3> </td> <td class="pagenavfirst"><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Biotechnology &amp; Viral Diagnostics','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#biotechnology-viral" class="">Bacterial Transformation</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=pGLO Bacterial Transformation','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#pglo" class="">pGLO Plasmid</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=CRISPR Gene Editing','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#crispr" class="">Gene Regulation</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=GFP','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#gfp" class="">Green Fluorescent Protein</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=PCR &amp; Real-Time PCR','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#pcr" class="">Other Resources</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Photosynthesis and Cellular Respiration','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#kit" class="">pGLO Bacterial Transformation and GFP Kits</a></td> <!---<td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Just for Fun','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#fun"><span class="forcebreaks">Just for Fun</span></a></td> <td class="pagenavlast"><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Posters for your Classroom','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#posters">Classroom Posters</a></td>---></tr> </tbody> </table> </div> <div class="opt-module opt-module-noborder opt-bg-blk"><img class="right" src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/GFP-pglo-jellyfish.png" alt="Green Fluorescent Protein (GFP) isolated from jellyfish" /> <p>Green fluorescent protein (GFP) is a protein that glows with a bright green fluorescence under ultraviolet light. First isolated from the marine jellyfish <em>Aequorea victoria</em>, the gene encoding GFP is used in cellular and molecular biology as a reporter to detect gene expression in transgenic organisms.</p> <p><a href="../category/pglo-plasmid-gfp-kits?ID=f75948d2-dc20-4a32-b4e5-b7e0fe4c21ed"><strong>Bio-Rad Explorer pGLO Plasmid and GFP Kits</strong></a> use the pGLO plasmid, which contains the <em>GFP</em> gene, to enable hands-on learning about the central dogma, gene expression and regulation, bacterial transformation, protein separation, and the biomanufacturing process.</p> </div> <div><a name="biotechnology-viral"></a></div> <div class="opt-module bg-white nobttm-brdr"> <h2>Bacterial Transformation</h2> <p>With pGLO bacterial transformation, students learn about genetic engineering as they transform a non-virulent laboratory strain of <em>Escherichia coli</em> (<em>E. coli</em>) with the pGLO plasmid. The procedure involves the CaCl<sub>2</sub>/heat shock method, which is a standard technique used in many research and biomanufacturing laboratories.</p> <ul class="list-set list-two-col list-set-th top-margin"> <li class="linkpointer-blank"><img src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/pglo-transformation-ppt.jpg" alt="pGLO Bacterial Transformation Powerpoint" /> <h3><span class="txt-f60"><a class="no-target-style" href="/webroot/web/software/lse/Downloads/pGLO_Transformation.ppt" target="_blank" rel="noopener noreferrer">pGLO Bacterial Transformation Powerpoint</a>&nbsp;</span><span class="filesize forcebreaks">(PPT 1.5 MB)</span></h3> <p>This editable presentation provides an overview of bacterial transformation with the pGLO plasmid.</p> </li> <li class="linkpointer-blank"><img src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/pglo-transformation.jpg" alt="pGLO Bacterial Transformation Video" /> <h3><span class="txt-f60"><a href="https://www.youtube.com/watch?v=DHVlSDXufjc" target="_blank" rel="noopener noreferrer" class="new-target-image" title="Link opens in new window">How To Perform a Bacterial Transformation Video</a></span></h3> <p>This video demonstrates how to transform bacteria using the Bio-Rad Explorer pGLO Bacterial Transformation Kit.</p> </li> </ul> </div> <div><a name="pglo"></a></div> <div class="opt-module optprodnav-bg nobttm-brdr"> <h2 class="nextp">pGLO Plasmid</h2> <a href="/webroot/web/pdf/lse/misc/pGLO_plasmid_sequence.txt" target="_blank" class="no-target-style" rel="noopener noreferrer"></a> <div class="right lt-margin20 width35"><a href="/webroot/web/pdf/lse/misc/pGLO_plasmid_sequence.txt" target="_blank" class="no-target-style" rel="noopener noreferrer"></a><center><a href="/webroot/web/pdf/lse/misc/pGLO_plasmid_sequence.txt" target="_blank" class="no-target-style" rel="noopener noreferrer"><img src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/pglo-map-sequence.jpg" alt="pGLO Sequence" style="width: 300px;" /></a><a class="bttn-block topmargin-1em no-target-style" href="/webroot/web/pdf/lse/misc/pGLO_plasmid_sequence.txt" target="_blank" rel="noopener noreferrer"><span class="bttn-orange" aria-label="Download the pGLO Sequence">Download the pGLO Sequence</span></a></center></div> <p>Bio-Rad&rsquo;s pGLO plasmid contains DNA sequences that enable its replication and expression of the fluorescent trait (phenotype) in bacteria following transformation. The essential sequences include the following:</p> <ul> <li><strong><em>GFP</em></strong> &mdash; jellyfish gene that encodes green fluorescent protein (GFP)</li> <li><strong>ori</strong> &mdash; origin of pGLO plasmid DNA replication (essential for making more copies of the plasmid)</li> <li><strong><em>bla</em></strong> &mdash; gene that encodes &beta;-lactamase, an enzyme that breaks down the antibiotic ampicillin; transformants expressing the <em>bla</em> gene can be selected by placing ampicillin in the growth medium</li> <li><strong>pBAD promoter</strong> &mdash; binds AraC-arabinose and promotes RNA polymerase binding and transcription of the <em>GFP</em> gene</li> <li><strong><em>araC</em></strong> &mdash; gene that encodes the regulatory protein that binds to the pBAD promoter; only when arabinose binds to the AraC protein is the production of GFP switched on</li> <li><strong>Multiple cloning site</strong> &mdash; a region containing restriction sites (NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion or deletion of a gene of interest</li> </ul> <p>Bacteria transformed with the pGLO plasmid are selected by ampicillin resistance and when induced to express GFP, they glow fluorescent green under UV light!</p> </div> <div><a name="pglo"></a></div> <div><a name="crispr"></a></div> <div class="opt-module nobttm-brdr"> <h2>Gene Regulation</h2> <p>Gene expression is carefully regulated to allow organisms to adapt to differing conditions and prevent wasteful production of proteins. Regulation often occurs at the level of transcription from DNA into RNA, specifically at the promoter, where RNA polymerase binds the DNA and begins transcription of the gene.</p> <p>In bacteria, groups of related genes are often clustered together and transcribed into RNA from one promoter. These clusters of genes controlled by a single promoter are called operons. The bacterial genes encoding the enzymes needed to metabolize the simple sugar arabinose are a perfect example. Three genes that encode the digestive enzymes involved in breaking down arabinose (<em>araB</em>, <em>araA</em>, and <em>araD</em>) are clustered together in arabinose operon 3, and all depend on initiation of transcription from a single promoter, pBAD. Transcription requires the simultaneous presence of RNA polymerase, a DNA-binding protein called AraC, and arabinose.</p> <ul> <li>When arabinose is absent, the AraC protein binds to the DNA at the binding site for RNA polymerase, preventing transcription of the digestive enzymes</li> <li>When arabinose is present, it interacts with AraC, causing AraC to change shape, allowing RNA polymerase to bind the promoter; <em>araB</em>, <em>araA</em>, and <em>araD</em> are then expressed and can do their job to break down arabinose until the arabinose runs out</li> </ul> <p>The pGLO plasmid contains both the promoter (pBAD) and <em>araC</em> gene, but <em>araB</em>, <em>araA</em>, and <em>araD</em> have been replaced by the single gene that codes for GFP, which serves as a reporter gene. In the presence of arabinose, the AraC protein promotes the binding of RNA polymerase to the promoter, which causes transcription of the <em>GFP</em> gene into messenger RNA (mRNA), followed by the translation of this mRNA into GFP. This process is called gene expression.</p> <p>As they produce more and more protein, the cells expressing GFP fluoresce a brilliant green. In the absence of arabinose, however, AraC no longer facilitates the binding of RNA polymerase, and the <em>GFP</em> gene is not expressed, and bacterial colonies have a wild-type (natural) phenotype &mdash; white colonies with no fluorescence.</p> <p></p> <p>This is an excellent example of the central dogma of molecular biology in action: <strong>DNA &gt; RNA &gt; protein &gt; trait</strong>.</p> <center><a href="/webroot/web/software/lse/Downloads/Bring_Inquiry_into_Your_Classroom_with_the_pGLO_Plasmid.ppt"><img src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/pglo-results.png" alt="Results of a pGLO bacterial transformation experiment" /></a></center> <p class="caption"><strong>Results of a pGLO bacterial transformation experiment.</strong> Controls that were incubated with no plasmid (-pGLO) grow as a lawn in the absence of ampicillin (LB plate) and do not grow at all in the presence of ampicillin (LB/amp). Transformants grown on ampicillin (LB/amp) grow as colonies but do not show GFP fluorescence; those grown in the presence of both amp and ara (LB/amp/ara) do glow green under UV light.</p> </div> <div><a name="gfp"></a></div> <div class="opt-module bg-f0f9f8 nobttm-brdr"> <h2>Green Fluorescent Protein</h2> <div class="right width30"><center><img alt="Green Fluorescent Protein (GFP) barrel structure" src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/GFP-th.jpg" style="width: 275px; height: 160px;" /></center> <div class="centered-div"> <p class="caption topmargin-1em">GFP has a barrel structure surrounding a central alpha helix that contains the fluorophore. It can be used as an example for discussions of protein secondary structure, parallel and anti-parallel beta sheets, and the use of genes and proteins in biotechnology. For more information, visit our partner <a href="https://www.3dmoleculardesigns.com/SearchResults.htm?Search_Keywords=gfp" target="_blank" rel="noopener noreferrer" class="new-target-image"><strong>3-D Molecular Designs</strong></a>.</p> </div> <br /><center><img style="right;width: 275px; height: 160px;" alt="GFP Purification with Chromatography and Electrophoresis" src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/GFP-electrophoresis-purification.jpg" /></center> <p class="topmargin-1em"><a href="/webroot/web/software/lse/Downloads/pGLO_GFP_Purification_Electrophoresis_and_Chromatography.ppt"><strong>GFP Purification &mdash; Electrophoresis and Chromatography</strong></a><span class="filesize forcebreaks"> (PPT 9.63 MB)</span></p> </div> <p>The GFP expressed from the pGLO plasmid illustrates the central doctrine of biology, from the transformation of DNA to the expression of a protein to the visualization of a trait. The bacterial proteome contains thousands of proteins, but only the cloned GFP glows!</p> <p>In its native environment, GFP fluoresces in the deep sea jellyfish, <em>Aequorea victoria</em>. Incredibly, GFP retains its fluorescent properties when cloned and expressed in <em>E. coli</em> and even when isolated from <em>E. coli</em> and separated on polyacrylamide gels or by chromatography. These amazing properties of GFP allow students to visualize the phenotypic properties of a protein and identify the single protein &ldquo;band&rdquo; responsible for the trait. These extensions link two of the most commonly used techniques in biotechnology labs: transformation and protein purification.</p> <p>Purification of a protein depends on a its chemical or physical properties, such as molecular weight, electrical charge, or solubility. GFP can be separated from others by its size using electrophoresis, and it is extremely hydrophobic, which enables its purification using hydrophobic interaction chromatography (HIC). When placed in a buffer containing a high concentration of salt, the HIC matrix selectively binds hydrophobic GFP molecules while allowing the bacterial proteins to pass right through the column. Then, simply lowering the salt concentration of the buffer causes GFP to elute from the column in a purer form.</p> <p>Students can explore these separation techniques by growing transformed bacteria in liquid culture to grow overnight, then lysing the cells to release their contents. The unique fluorescent property of GFP allows real-time monitoring of extraction and purification, modeling key processes used in biotechnology to produce and purify designer proteins with commercial or research value.</p> </div> <div><a name="pcr"></a></div> <div><a name="photosynthesis-cellular-respiration"></a></div> <div><a name="resources"></a></div> <div class="opt-module optresources-bg nobttm-brdr"> <h2>Other Resources</h2> <ul class="list-set list-two-col"> <li class="linkpointer-blank"><img src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/pglo-competency.jpg" alt="pGLO Bacterial Transformation Inquiries for Your Classroom" style="width: 400px; height: 222px;" /> <h3><span class="txt-f60"><a href="/webroot/web/software/lse/Downloads/Bring_Inquiry_into_Your_Classroom_with_the_pGLO_Plasmid.ppt" target="_blank" rel="noopener noreferrer" class="no-target-style" title="Link opens in new window">Bring Inquiry Into Your Classroom with the pGLO Plasmid</a>&nbsp;</span><span class="filesize forcebreaks">(PPT 9.06 MB)</span></h3> <p>Use pGLO Bacterial Transformation to illustrate the science and engineering practices described in the NGSS framework.</p> </li> <li class="linkpointer-blank"><img src="/webroot/web/images/lse/solutions/applications/preparing_for_class/classroom_resources/pglo-yt-channel-3col.jpg" alt="YouTube pGLO Bacterial Transformation Playlist" style="border: 1px solid #666; width: 400px;" /> <h3><a href="https://www.youtube.com/playlist?list=PL7_N-H8d6RiNkwLkza-ZhSdnl12lCXqXv" target="_blank" rel="noopener noreferrer" class="new-target-image" title="Link opens in new window"><span class="txt-f60">YouTube pGLO Bacterial Transformation Playlist</span></a></h3> <p>Use these short, instructional videos to enrich lessons about bacteria, bacterial transformation, and the green fluorescent protein (GFP).</p> </li> </ul> <div class="dotteddivider">&nbsp;</div> <h3 class="opt-subhead30 top-margin">Case Studies</h3> <p>Student-facing extensions that are also useful for AP exam prep.</p> <ul class="list-set list-two-col list-set-th top-margin"> <li class="linkpointer-blank"><img src="/webroot/web/images/lse/solutions/applications/preparing_for_class/classroom_resources/pglo-malaria-case-study-th.jpg" alt="Case Study: A Role for Bacterial Transformation in Controlling Malaria Transmission" /> <h3><a href="/webroot/web/pdf/lse/literature/10048977.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><span class="txt-f60">Case Study: A Role for Bacterial Transformation in Controlling Malaria Transmission</span></a> <span class="filesize forcebreaks">(PDF 3.3 MB)</span></h3> <p></p> </li> <li class="linkpointer-blank"><img src="/webroot/web/images/lse/solutions/applications/preparing_for_class/classroom_resources/pglo-gut-microbiome-case-study-th.jpg" alt="Case Study: Hacking the Gut Microbiome" /> <h3><a class="no-target-style" href="https://info.bio-rad.com/rs/272-THL-329/images/12003388.pdf" target="_blank" rel="noopener noreferrer"><span class="txt-f60">Case Study: Hacking the Gut Microbiome</span></a> <span class="filesize forcebreaks">(PDF 1.5 MB)</span></h3> <p></p> </li> </ul> <table class="right width40" style="text-align: center;" border="0" width="400" role="presentation"></table> <script type="text/javascript" src="/webroot/web/js/apptech-opt-conveter-min.js"></script> <a class="scrollup" href="#" style="display: none;">Scroll</a></div> <div><a name="pglo"></a></div> <div><a name="kit"></a></div> <div class="opt-module nobttm-brdr"> <h2>pGLO Bacterial Transformation and GFP Kits</h2> <img class="right" src="/webroot/web/images/lse/products/pglo_gfp_kits/category_feature/global/pglo-bacterial-transformation-kit.jpg" alt="pGLO Bacterial Transformation Kit" style="width: 350px;" /> <p>pGLO Lab Kits utilize Bio-Rad&rsquo;s pGLO plasmid, which encodes a green fluorescent protein (GFP), to enable instructors to give students a hands-on introduction to transformation, cloning, protein chromatography, and electrophoresis techniques.</p> <p></p> <h3><a href="../product/pglo-bacterial-transformation-kit?ID=619b8f74-9d3f-4c2f-a795-8a27e67598b7&amp;WT.mc_id=191014027131">pGLO Bacterial Transformation Kit</a></h3> <p>Use bacterial transformation with an inducible promoter to make glowing<br /><em>E. coli</em>.</p> <h3><a href="../product/pglo-transformation-inquiry-kit-for-ap-biology?ID=NEXYOKKG4&amp;WT.mc_id=191014027131">pGLO Transformation and Inquiry Kit for AP Biology</a></h3> <p>Investigate the functional elements of pGLO bacterial transformation, including heat shock, antibiotic selection, promoters, and satellite colony formation.</p> <h3><a href="../product/pglo-sds-page-extension?ID=a41608e9-b348-43e0-98bb-d0ae12664e06">pGLO SDS-PAGE Extension</a></h3> <p>Use protein electrophoresis to view the expression of proteins in your pGLO bacteria.</p> <h3><a href="../product/green-fluorescent-protein-chromatography-kit?ID=7f20553c-73f8-498a-b0b1-75d985308bf8">Green Fluorescent Protein Chromatography Kit</a></h3> <p>Use chromatography to purify glowing green fluorescent protein from your pGLO bacteria.</p> <h3><a href="../product/secrets-rainforest-kit?ID=6b225c3a-5f77-4680-8b34-1361fecf8f4e">Secrets of the Rainforest Kit</a></h3> <p>Simulate the drug discovery process in your classroom with a glowing protein.</p> </div> 1
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