Difference between revisions of "Team:Freiburg/Project/Coli Strains"

 
(10 intermediate revisions by 7 users not shown)
Line 2: Line 2:
 
{{Freiburg/Menubar}}
 
{{Freiburg/Menubar}}
  
{{Freiburg/wiki_content_start}}
+
{{Team:Freiburg/wiki_content_start_bubble}}
 
<html>
 
<html>
<!-- Labjournal content goes in here -->
+
<style>
+
/*========= BEGIN: style for navigation bar ==========*/
<div class="content_box">
+
#notebook {
<h3 class="sectionedit1">Information about viruses and bacteria</h3>
+
    background: url(https://static.igem.org/mediawiki/2015/c/cd/Freiburg_icon_notebook_active_yellow.png) no-repeat;
<div class="level3">
+
}
</div>
+
<h5>German Measles</h5>
+
<div class="level5">
+
<p>
+
The Rubella Virus (RV) is the only member of the genus <em>Rubiviridae</em> and belongs to the family of <em>Togaviridae</em>. It contains single-stranded plus-sense RNA, which encodes three structural and two non-structural proteins <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup>.  Two of the structural proteins are the envelope proteins E1 and E2 that form heterodimers arranged in groups of three and are distributed all over the viruses surface <sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>. As the glycoprotein E1 appears to be immunodominant <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a></sup>, we decided to use it with the respective sequence as RV antigen for the DiaCHIP and let it be synthesized by IDT. <br/>
+
  
The Rubella Virus is transmitted via airborne infection with humans as the only known hosts. It causes the rubella disease, also known as German measles or Rubeola. After the first infection the virus persists in the body for the whole life providing lifelong immunity and the diseases can therefore be referred to as typical for childhood. It goes along with exanthemas, fever, headache, rheumatic pains and swollen lymph nodes. The infection can anyways for some patients be asymptomatic as well. In contrast, in rare cases it can come to complications. Those are more frequent if the patient already reached adulthood. In such cases arthritis, bronchitis, encephalitis and inclusion of heart damages are conceivable <sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup>. <br/>
+
#notebook a {
 +
    color: #ecdc18;
 +
}
  
As RV can also cross the placenta, pregnant women getting infected in the beginning of their pregnancy might lose their child due to a spontaneous abort. The children can also be born but in many cases suffer from congenital rubella syndromes  <sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a></sup> such as deformities of the heart, cataracts or labyrinthine deafness.
+
#runningchip {
Therapies are only symptomatic but there is vaccination available that could prevent the disease what is especially recommended to women planning to get pregnant.<br/>
+
    left: 85.5%;
</p>
+
/*========= END: style for navigation bar ==========*/
</div>
+
</style>
<h5>Herpes Simplex</h5>
+
<div class="level5">
+
<p>
+
A lot of people might have already had little blisters in the area of the lip that disappeared after some days. In most cases those might have been provoked by the Herpes Simplex Virus Type 1 (HSV1).<br/>
+
  
HSV1 belongs to the Human Herpesviruses, as well as the Varicella Zoster Virus, and is therefore part of the family of <em>Herpesviridae</em>. It contains double-stranded DNA and an envelope consisting of at least 10 viral proteins surrounds the capsid. Most of these proteins are glycoproteins, one of which is the glycoprotein G that we express as HSV antigen. It is the glycoprotein differing the most among species so it allows their discrimination <sup><a class="fn_top" href="#fn__6" id="fnt__6" name="fnt__6">6)</a></sup>. Glycoprotein G is important for the attachment to the cell and the entry of the virus into it <sup><a class="fn_top" href="#fn__7" id="fnt__7" name="fnt__7">7)</a></sup>.<br/>
+
<script type="text/javascript">
 +
//===================BEGIN:Amazing Bubble Sidebar==========================
  
There are two different types of Herpes Simplex Virus with about 99% of identity in the gene regions. Both types are transmitted via the contact of mucous membranes. Type 1 – the one we are working with – can be transmitted via kissing, type 2 is mainly transmitted via sexual contact. Below we are only referring to the Herpes Simplex Virus Type 1. It remains in the body in a latent state and several outbreaks during life are possible. As about 85-90% of the world’s population is seropositive the primary infection often takes place prior to the age of five due to a transmission from the parents to their child <sup><a class="fn_top" href="#fn__8" id="fnt__8" name="fnt__8">8)</a></sup>. The infection of newborns is dangerous to life.<br/>
+
$(document).ready(function(){
 +
  // CHANGE THE FOLLOWING ATTRIBUTES //
 +
  var href_text1='https://2015.igem.org/Team:Freiburg/Notebook',
 +
  // Text2 needs no href as it is the actual page //
 +
  img_url='https://static.igem.org/mediawiki/2015/9/99/Freibur_icon_notebook_white_03.png',
 +
  href_text3='https://2015.igem.org/Team:Freiburg/Protocols',
 +
  // Text1 needs no text as it is a pic //
 +
  text2='E.coli Strains';
 +
  // Text3 needs no text as its always 'next' //
 +
  // HOLD ON CHANGING THINGS --JABBERWOCK  //
  
After intruding the body the virus proliferates and is shed via the mucous membrane. It also infiltrates neurons in which it persists lifelong, as already mentioned. In very severe cases the virus can cause encephalitis as well as meningitis. The reasons for sudden outbreaks of the virus are not clearly understood. A suppression of the immune system or stress are only some possible explanations.<br/>
+
  $('#bubble1').attr('href',href_text1);
 +
  $('#bubble1_img').attr('src', img_url);
 +
  $('#bubble3').attr('href',href_text3);
  
A therapy with anti-viral drugs is possible, for cutaneous infections in facial or rather labial areas the application of corticoids is however usually sufficient <sup><a class="fn_top" href="#fn__9" id="fnt__9" name="fnt__9">9)</a></sup>.<br/>
+
  $('#bubble2').text(text2);
</p>
+
});
</div>
+
<h5>Tetanus</h5>
+
<div class="level5">
+
<p>
+
If the ambulance is called to take care of an injured person often the first thing they do is to perform a preventing vaccination against <em>Clostridium tetani</em>. This is a little anaerobic gram-positive rod-shaped bacillus that forms spores. These can even survive in inhospitable areas and are present in soil. They can enter the human body easily via small wounds which is why injured people are at high risk of getting infected with <em>C. tetani</em> but cannot transmit the infection to other individuals.<br/>
+
  
In regions where people are not vaccinated and good health care is not provided, tetanus, which is caused by the bacterium, is a very common cause of death following injuries. The bacterium produces two toxins, named tetanospasmin and tetanolysin. The former was found to cause tetanus by reaching the bone marrow via the nerves. There it is responsible for provoking hypersensitivity, increased reflexes and spasms <sup><a class="fn_top" href="#fn__10" id="fnt__10" name="fnt__10">10)</a></sup>. The latter causes damage to the heart muscle and blood components <sup><a class="fn_top" href="#fn__11" id="fnt__11" name="fnt__11">11)</a></sup>.<br/>
+
//===================END:Amazing Bubble Sidebar==========================
 +
</script>
 +
<style type="text/css">
 +
 +
.image_box.left {
 +
width: 200px;
 +
}
  
For our DiaCHIP we expressed a part of the tetanospasmin protein that is generally referred to as tetanustoxin. It consists of a heavy and a light chain that are linked by disulfide bonds <sup><a class="fn_top" href="#fn__12" id="fnt__12" name="fnt__12">12)</a></sup>. We worked with the carboxyl-terminal domain of the heavy chain that is able to bind to the target membrane and allows the internalization of the actual toxic region of the light chain <sup><a class="fn_top" href="#fn__13" id="fnt__13" name="fnt__13">13)</a></sup>. As we did not express any of the toxic fragments there was no need for special safety measures. The toxin fragment interfering with the neuronal system was not expressed and the part we used is not able to reproduce itself.<br/>
+
</style>
 +
 +
<div class="content_box">
 +
<h1 class="sectionedit1">E.coli genotypes</h1>
 +
<div class="level1">
 +
<p>
 +
We are using <i>E.coli</i> strains for overexpression of our target proteins. <i>E. coli</i> is suitable for expression of a wide range of heterologous proteins. Our aim was the enrichment of active, soluble recombinant protein. Therefore, we used different <i>E.coli</i> strains and adapted different expression temperatures, durations and IPTG concentrations to get as much soluble target protein as possible.
 +
 
 +
Of course, many challenges can arise during expression of recombinant proteins. That is why most expression strains have a few mutations in common to obtain a high yield of expressed proteins.
  
After an infection with <em>C. tetani</em> the first symptoms are headache, muscle and dorsal pain and a feeling of being tired. Additionally, the patient may feel some tautness in the area of the injury and reveal sensitivity to light and noise. If the patient is not taken care of, the infection is manifested by local stiffening of muscles, mainly those in the area of the jaw and neck. In the following progression the patient will suffer from high fever and muscle spasms. Those will at first be located in the face but spread over the whole body what causes the typical extended position. The immense tension in the muscles due to the effect of the toxins can cause lesions as well as dislocations of the joints or broken bones. As a consequence, many patients suffer from shortened muscles, ankylosis (stiffness of the joints) or spine deformity. In case of the lack of appropriate health care, death due to suffocation or cardiovascular failure is common and occurs partially despite previous vaccination.<br/>
+
Expression strains often harbor mutations that make them deficient in outer membrane protease VII and delete the Ion protease. Both of this reduces proteolysis of the expressed recombinant proteins. Additionally the native restriction/methylation system is often inactivated <sup><a class="fn_top" href="#fn__1" id="fnt__1" name="fnt__1">1)</a></sup><sup><a class="fn_top" href="#fn__2" id="fnt__2" name="fnt__2">2)</a></sup>.
  
To avoid long-term effects due to an infection with <em>C. tetani</em> or even death, the wound is excised and surgically taken care of. Additionally, the patient will be treated with antibiotics and will be given antibodies targeting the toxin.<br/>
+
Below we introduce the bacterial strains we used for protein overexpression:
 
</p>
 
</p>
 
</div>
 
</div>
<h5>Varicella Zoster / Herpes Zoster</h5>
+
<!-- EDIT1 SECTION "E.coli genotypes" [1-1164] -->
<div class="level5">
+
<h2 class="sectionedit2">Bl21 (DE3) pLysS</h2>
 +
<div class="level2">
 +
<div class="image_box left">
 +
<a class="media" href="https://static.igem.org/mediawiki/2015/8/87/Freiburg_files-bl21.png" title="files:bl21.png"><img align="left" alt="" class="medialeft" src="https://static.igem.org/mediawiki/2015/8/87/Freiburg_files-bl21.png" width="150"/></a>
 +
</div>
 
<p>
 
<p>
An infection going along with red and itching skin that nearly every person in the world suffers from…most might know it as chickenpox and might not even remember the first infection as this often takes place during childhood. The infection is caused by the Varicella Zoster Virus (VZV) that belongs to the family of <em>Herpesviridae</em>, the same family as the Herpes Simplex Virus, and contains double-stranded DNA surrounded by a capsid. A tegument fills the space between this capsid and the envelope <sup><a class="fn_top" href="#fn__14" id="fnt__14" name="fnt__14">14)</a></sup>. This outer layer contains different viral envelope glycoproteins, one of which is the glycorprotein E we used for the DiaCHIP as VZV antigen <sup><a class="fn_top" href="#fn__15" id="fnt__15" name="fnt__15">15)</a></sup>. It forms heterodimers with the glycoprotein I and was found to play an important role in cell-cell attachment as well as facilitating the entry of the virus and the assembly of the virion <sup><a class="fn_top" href="#fn__16" id="fnt__16" name="fnt__16">16)</a></sup>.<br/>
+
BL21 (DE3) is the basic IPTG-inducible strain containing T7 RNA-polymerase (DE3) for general protein expression <sup><a class="fn_top" href="#fn__3" id="fnt__3" name="fnt__3">3)</a></sup>. The <em>E.coli</em> expression strain BL21 (DE3) pLysS is useful for the expression of toxic proteins.
  
VZV is transmitted via droplet infection or by having contact with blisters or mucous membranes. Following the first contact with the virus and with an incubation time of about two weeks the patient suffers from fever and exanthemas that normally heal without leaving scars. This first infection is called Varicella Zoster or, as mentioned above, chickenpox. In the United States for example more than 95 percent of persons older than 20 years were seropositive for VZV <sup><a class="fn_top" href="#fn__17" id="fnt__17" name="fnt__17">17)</a></sup>.<br/>
+
The pLysS plasmid contains a chloramphenicol resistance. The pLysS plasmid also encodes T7 phage lysozyme, an inhibitor for T7 polymerase. In the absence of IPTG induction this inhibitor reduces and almost eliminates expression from a transformed T7 promoter containing plasmids .
 
+
This inducable expression is suitable to stabilize recombinants encoding particularly toxic proteins <sup><a class="fn_top" href="#fn__4" id="fnt__4" name="fnt__4">4)</a></sup><sup><a class="fn_top" href="#fn__5" id="fnt__5" name="fnt__5">5)</a></sup>.  
As the virus resides in some ganglions of the body, mostly elderly or immune deficient people might again be afflicted with the disease that is then called Herpes Zoster. It manifests itself in exanthemas restricted to the area of the respective ganglion that is affected. Additionally, the patients are very sensitive to skin contact, have a fever and feel pain. In some cases the reactivation of the virus can lead to neuritis.
+
</p>
Normally the infection with the virus, the primary infection as well as the reactivation of the virus, are not life threatening and in most cases end without consequences for the patient. The primary infection is only a threat for newborns and immune deficient patients where a hemorrhagic development can be lethal. If adults are for the first time exposed to the virus it can also cause much more severe damage. The development of defects in the central nervous system is one of the most prominent consequences <sup><a class="fn_top" href="#fn__18" id="fnt__18" name="fnt__18">18)</a></sup>.<br/>
+
</div>
 +
<!-- EDIT2 SECTION "Bl21 (DE3) pLysS" [1165-2091] -->
 +
<h2 class="sectionedit3">Rosetta(DE3)pLysS</h2>
 +
<div class="level2">
 +
<div class="image_box left">
 +
<a class="media" href="https://static.igem.org/mediawiki/2015/1/18/Freiburg_files-rosetta.png" title="files:rosetta.png"><img align="left" alt="" class="medialeft" src="https://static.igem.org/mediawiki/2015/1/18/Freiburg_files-rosetta.png" width="200"/></a>
 +
</div>
  
There is a live attenuated varicella vaccine available that is mainly applied for children and risk patients. Passive immunization with IgG antibodies is often the choice for exposed pregnant women and newborns within a time span of about 48 hours <sup><a class="fn_top" href="#fn__19" id="fnt__19" name="fnt__19">19)</a></sup>.
+
<p>
 +
Rosetta™ host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in <i>E. coli</i>. These strains supply tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid. The Rosetta strains provide a better translation for eukaryotic proteins that have not been codon optimized. In Rosetta(DE3)pLysS, the rare tRNA genes are present on the same plasmid that carries the T7 lysozyme gene for inducable expression <sup><a class="fn_top" href="#fn__6" id="fnt__6" name="fnt__6">6)</a></sup>.
 
</p>
 
</p>
 
</div>
 
</div>
<h5>Syphilis</h5>
+
<!-- EDIT3 SECTION "Rosetta(DE3)pLysS" [2092-2709] -->
<div class="level5">
+
<h2 class="sectionedit4">C43 (DE3)</h2>
 +
<div class="level2">
 +
<div class="image_box left">
 +
<a class="media" href="https://static.igem.org/mediawiki/2015/5/58/Freiburg_files-c43.png" title="files:c43.png"><img align="left" alt="" class="medialeft" src="https://static.igem.org/mediawiki/2015/5/58/Freiburg_files-c43.png" width="150"/></a>
 +
</div>
 +
<p>
 +
This overexpression strain derives from the C41 (DE3) <i>E.coli</i> strain.
 +
These strains contain an uncharacterized genetic mutation that allows a higher tolerance to toxic proteins. The mutation prevents <i>E.coli</i> cell death often associated with expression of many recombinant toxic proteins.
 +
</p>
 +
<p>
 +
The advantage of this strain to BL21 (DE3) is that even membrane proteins, often particularly problematic for protein expression, can be overexpressed <sup><a class="fn_top" href="#fn__7" id="fnt__7" name="fnt__7">7)</a></sup>.
 +
</p>
 +
<p>
 +
Received from the Toolbox - BIOSS (Centre for Biological Signalling Studies).
 +
</p>
 +
</div>
 +
<!-- EDIT4 SECTION "C43 (DE3)" [2710-3520] -->
 +
<h2 class="sectionedit5">ArcticExpress (DE3)RP</h2>
 +
<div class="level2">
 +
<div class="image_box left"
 +
<a class="media" href="https://static.igem.org/mediawiki/2015/b/ba/Freiburg_files-arctic.png" title="files:arctic.png"><img align="left" alt="" class="medialeft" src="https://static.igem.org/mediawiki/2015/b/ba/Freiburg_files-arctic.png" width="125"/></a>
 +
</div>
 +
<p>
 +
ArcticExpress Competent Cells are engineered to address the common bacterial gene expression hurdle of protein insolubility. These cells are derived from the high-performance Stratagene BL21-Gold competent cells, enabling efficient high-level expression of heterologous proteins in <i>E. coli</i>. With these cells we were able to express our target proteins o/n at 10°C. Low-temperature cultivation represents one strategy for increasing the recovery of soluble protein. This is due to the fact that <i>E. coli</i> chaperonins, which facilitate proper protein folding by binding and stabilizing unfolded or partially folded proteins, lose activity at reduced temperatures <sup><a class="fn_top" href="#fn__8" id="fnt__8" name="fnt__8">8)</a></sup><sup><a class="fn_top" href="#fn__9" id="fnt__9" name="fnt__9">9)</a></sup><sup><a class="fn_top" href="#fn__10" id="fnt__10" name="fnt__10">10)</a></sup>.
 +
</p>
 +
<p>
 +
Received from AG Weber - BIOSS (Centre of Biological Signalling Studies).
 +
</p>
 +
</div>
 +
<!-- EDIT5 SECTION "ArcticExpress (DE3)RP" [3521-4579] -->
 +
<h2 class="sectionedit6">Top10</h2>
 +
<div class="level2">
 +
<div class="image_box left"
 +
<a class="media" href="https://static.igem.org/mediawiki/2015/0/0f/Freiburg_files-top10.png" title="files:top10.png"><img align="left" alt="" class="medialeft" src="https://static.igem.org/mediawiki/2015/0/0f/Freiburg_files-top10.png" width="200"/></a>
 +
</div>
 
<p>
 
<p>
One of the most common sexually transmittable diseases nowadays, besides AIDS, is Syphilis that is also a chronical disease. It is caused by the bacterium <em>Treponema pallidum</em> subsp. <em>pallidum</em>, which is a gram-negative member of the family of four <em>Spirochaetaceae</em> and exhibits a spiral shape <sup><a class="fn_top" href="#fn__20" id="fnt__20" name="fnt__20">20)</a></sup>.<br/>
+
The Top10 cloning strain harbors some mutations especially useful for high efficiency cloning purposes and for propagation of plasmids.
<em>T. pallidum</em> has a rather small genome as it lacks many coding regions e.g. for metabolic enzymes. Nonetheless, its gene encodes the bacterioferritin protein TpF1 that was found to be immunogenic and useful for serodiagnosis of different stages of syphilis <sup><a class="fn_top" href="#fn__21" id="fnt__21" name="fnt__21">21)</a></sup>. This is why we used this recombinant protein as <em>T. palldium</em> antigen for the DiaCHIP.<br/>
+
In this strain, the gene for the type I endonuclease complex KI that recognizes unmethylated DNA is not functional <sup><a class="fn_top" href="#fn__11" id="fnt__11" name="fnt__11">11)</a></sup>. So genes from PCR amplification are not degraded by this defense-system and can be efficiently transformed into the bacteria.<br/>
  
The bacterium is very difficult to cultivate <em>in vitro</em>, partly due to the absence of some metabolic capabilities. Humans are the only natural hosts of <em>T. pallidum</em> and can be passed from one individual to another not only during sex but also via kissing if there are small lesions in the mucous membrane through which they can escape and enter <sup><a class="fn_top" href="#fn__22" id="fnt__22" name="fnt__22">22)</a></sup>.<br/>
+
Additionally, a mutation in a nonspecific endonuclease (endA) yields cleaner preparations due to less unspecifically cut double-strand DNA <sup><a class="fn_top" href="#fn__12" id="fnt__12" name="fnt__12">12)</a></sup>.<br/>
  
After infection with <em>T. pallidum</em> there are four stages of syphilis distinguishable. Primary syphilis is characterized by chancre at the place of infiltration but normally heals during a time span of three to 7 weeks. Additionally, in this first phase the patient might suffer from swollen lymph nodes.<br/>
+
Finally the rate of homologous recombination that can hinder efficient cloning is reduced by a mutation in the DNA strand exchange and recombination protein recA<sup><a class="fn_top" href="#fn__13" id="fnt__13" name="fnt__13">13)</a></sup>.
 
+
The secondary syphilis manifests itself in exanthemas and moistening areas of the skin, mainly in the genital area and between fingers and toes, which are highly infectious. There might also show up enanthemas on mucous membranes and the patient might lose its hair.<br/>
+
 
+
The symptoms of these first two stages of syphilis often fade after about three months and a latency period of variable time, up to years, may follow. The patient is still contagious in the early time of this period but loses this characteristic after a while.<br/>
+
 
+
If the infection with <em>T. pallidum</em> reaches the next stage, inner organs are affected what may lead to hepatitis or damages involving the aorta for example. This is also the first stage in which the neuronal system is mostly battered. At latest a neurosyphilis develops during quaternary syphilis and the patient suffers from psychoses and the nerves shed their myelinisation.<br/>
+
 
+
It is also possible that organisms of <em>T. pallidum</em> are transmitted via the bloodstream from mother to child causing congenital syphilis. Depending on the immune response of the fetus consequences may vary from fetal death to fetal damage or an infected newborn. The defects can also have a wide range and affect growth, internal organs like liver or spleen and the neuronal system. The onset of these effects can start only after years but also immediately after birth <sup><a class="fn_top" href="#fn__23" id="fnt__23" name="fnt__23">23)</a></sup>.<br/>
+
 
+
If the disease is noticed early enough a treatment with penicillin can lead to a cure with, in best cases, no permanent harm <sup><a class="fn_top" href="#fn__24" id="fnt__24" name="fnt__24">24)</a></sup>.
+
 
</p>
 
</p>
 +
<div class="tags"><span>
 +
<a class="wikilink1" href="/igem2015/doku.php?id=tag:info&amp;do=showtag&amp;tag=info" rel="tag" title="tag:info">info</a>
 +
</span></div>
 
</div>
 
</div>
 +
<!-- EDIT6 SECTION "Top10" [4580-] --><div class="footnotes">
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup>
 +
    <a href="http://www.genetics.org/content/54/1/61.full.pdf+html;"  target="_blank">Demerec M. et al. (1966). A proposal for a uniform nomenclature in bacterial genetics. Genetics. 54(1):61-76</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__2" id="fn__2" name="fn__2">2)</a></sup>
 +
<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC210899/pdf/jbacter00181-0229.pdf;"  target="_blank">Grodberg, J. and Dunn, J. J. (1988) J Bacteriol 170(3):1245-53</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__3" id="fn__3" name="fn__3">3)</a></sup>
 +
<a href="http://www.sciencedirect.com/science/article/pii/0092867487905630"  target="_blank">Moffatt BA, Studier FW (1987). T7 lysozyme inhibits transcription by T7 RNA-polymerase. Cell. 49(2):221-7</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__4" id="fn__4" name="fn__4">4)</a></sup>
 +
<a href="https://www.agilent.com/cs/library/usermanuals/Public/230191.pdf"  target="_blank">Jerpseth, M., Jerpseth, B., Briester, L. and Greener, A. (1998) Strategies 11(1):3-4</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__5" id="fn__5" name="fn__5">5)</a></sup>
 +
<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731896/pdf/gkp498.pdf"  target="_blank">Jerpseth, B., Callahan, M. and Greener, A. (1997) Strategies10(2):37–38</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__6" id="fn__6" name="fn__6">6)</a></sup>
 +
<a href="http://nar.oxfordjournals.org/content/20/17/4668.full.pdf"  target="_blank">Looman et al. (1987) EMBO J. 6, 2489-2492</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__7" id="fn__7" name="fn__7">7)</a></sup>
 +
<a href="http://www.sciencedirect.com/science/article/pii/S002228369690399X"  target="_blank">B Miroux and J E Walker, ‘Over-Production of Proteins in Escherichia Coli: Mutant Hosts That Allow Synthesis of Some Membrane Proteins and Globular Proteins at High Levels.’, Journal of molecular biology, 260 (1996), 289–98</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__8" id="fn__8" name="fn__8">8)</a></sup>
 +
<a href="http://download.springer.com/static/pdf/195/art%253A10.1007%252Fs00253-015-6744-5.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs00253-015-6744-5&token2=exp=1442320220~acl=%2Fstatic%2Fpdf%2F195%2Fart%25253A10.1007%25252Fs00253-015-6744-5.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs00253-015-6744-5*~hmac=662f291fc8a55dcdbd8d5cea91926d4aa442bf111b9b8be2c8f048c364fe8b9d"  target="_blank">Carstens, C.-P., Bonnardel, J., Allen, R. and Waesche, A. (2001) Strategies 14:50</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__9" id="fn__9" name="fn__9">9)</a></sup>
 +
<a href="http://www.chem-agilent.com/pdf/strata/230193.pdf"  target="_blank">Schein, C. H. (1989) Biotechnology7:1141-8</a>
 +
</div>
 +
<div class="fn"><sup><a class="fn_bot" href="#fnt__10" id="fn__10" name="fn__10">10)</a></sup>
 +
<a href="http://www.nature.com/nbt/journal/v21/n11/full/nbt1103-1266.html"  target="_blank">Ferrer, M., Chernikova, T. N., Yakimov, M.M., Golyshin, P. N.and Timmis, K. N. (2003) Nat Biotechnol.21(11):1266-7</a>
 
</div>
 
</div>
<div class="content_box">
 
<!-- EDIT1 SECTION "Information about viruses and bacteria" [53-] --><div class="footnotes">
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__1" id="fn__1" name="fn__1">1)</a></sup>
 
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574052/" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574052/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574052/</a>|Abernathy et al.: Analysis of whole genome sequences of 16 strains of rubella virus from the United States, 1961-2009</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__2" id="fn__2" name="fn__2">2)</a></sup>
 
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457135/" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457135/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457135/</a>|Rossmann et al.: Cryo-Electron Tomography of Rubella Virus</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__3" id="fn__3" name="fn__3">3)</a></sup>
 
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC227930/pdf/330270.pdf" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC227930/pdf/330270.pdf">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC227930/pdf/330270.pdf</a>|Best et al.: Use of Rubella Virus E1 Fusion Proteins for Detection of Rubella Virus Antibodies</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__4" id="fn__4" name="fn__4">4)</a></sup>
 
<a class="urlextern" href="http://ac.els-cdn.com/S1744165X07000182/1-s2.0-S1744165X07000182-main.pdf?_tid=92ea6500-4e6f-11e5-89e9-00000aab0f26&amp;acdnat=1440867861_4325df64785c55431e4a3dcf9a9fce47" rel="nofollow" target="_Blank" title="http://ac.els-cdn.com/S1744165X07000182/1-s2.0-S1744165X07000182-main.pdf?_tid=92ea6500-4e6f-11e5-89e9-00000aab0f26&amp;acdnat=1440867861_4325df64785c55431e4a3dcf9a9fce47">http://ac.els-cdn.com/S1744165X07000182/1-s2.0-S1744165X07000182-main.pdf?_tid=92ea6500-4e6f-11e5-89e9-00000aab0f26&amp;acdnat=1440867861_4325df64785c55431e4a3dcf9a9fce47</a>|Best: Rubella</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__5" id="fn__5" name="fn__5">5)</a></sup>
 
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pubmed/9450233" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pubmed/9450233">http://www.ncbi.nlm.nih.gov/pubmed/9450233</a>|Frey: Neurological aspects of rubella virus infection</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__6" id="fn__6" name="fn__6">6)</a></sup>
 
<a class="urlextern" href="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073842" rel="nofollow" target="_Blank" title="http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073842">http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073842</a>|Han et al.: Development of Recombinant Antigen Array for Simultaneous Detection of Viral Antibodies</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__7" id="fn__7" name="fn__7">7)</a></sup>
 
<a class="urlextern" href="http://ac.els-cdn.com/S009286740081363X/1-s2.0-S009286740081363X-main.pdf?_tid=75b0f3ae-4f0b-11e5-ae49-00000aab0f6b&amp;acdnat=1440934814_43bc6e9632ca6586a27a3e28e0441da2" rel="nofollow" target="_Blank" title="http://ac.els-cdn.com/S009286740081363X/1-s2.0-S009286740081363X-main.pdf?_tid=75b0f3ae-4f0b-11e5-ae49-00000aab0f6b&amp;acdnat=1440934814_43bc6e9632ca6586a27a3e28e0441da2">http://ac.els-cdn.com/S009286740081363X/1-s2.0-S009286740081363X-main.pdf?_tid=75b0f3ae-4f0b-11e5-ae49-00000aab0f6b&amp;acdnat=1440934814_43bc6e9632ca6586a27a3e28e0441da2</a>|Montgomery et al.: Herpes Simplex Virus-1 Entry into Cells Mediated by a Novel Member of the TNF/NGF Receptor Family</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__8" id="fn__8" name="fn__8">8)</a></sup>
 
<a class="urlextern" href="http://download.springer.com/static/pdf/219/art%253A10.1007%252Fs11910-013-0414-8.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs11910-013-0414-8&amp;token2=exp=1440930898~acl=%2Fstatic%2Fpdf%2F219%2Fart%25253A10.1007%25252Fs11910-013-0414-8.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs11910-013-0414-8" rel="nofollow" target="_Blank" title="http://download.springer.com/static/pdf/219/art%253A10.1007%252Fs11910-013-0414-8.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs11910-013-0414-8&amp;token2=exp=1440930898~acl=%2Fstatic%2Fpdf%2F219%2Fart%25253A10.1007%25252Fs11910-013-0414-8.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs11910-013-0414-8">http://download.springer.com/static/pdf/219/art%253A10.1007%252Fs11910-013-0414-8.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs11910-013-0414-8&amp;token2=exp=1440930898~acl=%2Fstatic%2Fpdf%2F219%2Fart%25253A10.1007%25252Fs11910-013-0414-8.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs11910-013-0414-8</a>*~hmac=ceabec4cc066dc36aae263e7af9b7b420316b250b14dd49583f58eda330f4549|Steiner, Benninger: Update on Herpes Virus Infections of the Nervous System</div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__9" id="fn__9" name="fn__9">9)</a></sup>
 
<a class="urlextern" href="http://herpes.de/herpesformen/lippenherpes" rel="nofollow" target="_Blank" title="http://herpes.de/herpesformen/lippenherpes">http://herpes.de/herpesformen/lippenherpes</a></div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__10" id="fn__10" name="fn__10">10)</a></sup>
 
<a class="urlextern" href="http://annals.org/article.aspx?articleid=746855" rel="nofollow" target="_Blank" title="http://annals.org/article.aspx?articleid=746855">http://annals.org/article.aspx?articleid=746855</a>|Afshar et al.: Narrative Review: Tetanus – A Health Threat After Natural Disasters in Developing Countries</div>
 
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__11" id="fn__11" name="fn__11">11)</a></sup>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__11" id="fn__11" name="fn__11">11)</a></sup>
<a class="urlextern" href="http://symptomat.de/Clostridium_tetani" rel="nofollow" target="_Blank" title="http://symptomat.de/Clostridium_tetani">http://symptomat.de/Clostridium_tetani</a></div>
+
<a class="urlextern" href="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG10459" rel="nofollow" target="_Blank" title="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG10459">http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG10459</a></div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__12" id="fn__12" name="fn__12">12)</a></sup>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__12" id="fn__12" name="fn__12">12)</a></sup>
<a class="urlextern" href="http://www.jbc.org/content/280/51/42336.full.pdf+html" rel="nofollow" target="_Blank" title="http://www.jbc.org/content/280/51/42336.full.pdf+html">http://www.jbc.org/content/280/51/42336.full.pdf+html</a>|Bohner, Schiavo: Tetanus Toxin Is Transported in a Novel Neuronal Compartment Characterized by a Specialized pH Regulation</div>
+
<a class="urlextern" href="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG11336" rel="nofollow" target="_Blank" title="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG11336">http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG11336</a></div>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__13" id="fn__13" name="fn__13">13)</a></sup>
 
<div class="fn"><sup><a class="fn_bot" href="#fnt__13" id="fn__13" name="fn__13">13)</a></sup>
<a class="urlextern" href="http://ac.els-cdn.com/S0171298510001567/1-s2.0-S0171298510001567-main.pdf?_tid=05754f9e-4f34-11e5-95a1-00000aacb360&amp;acdnat=1440952235_9b35c0a0389b3dbe95d48a4e8a583234" rel="nofollow" target="_Blank" title="http://ac.els-cdn.com/S0171298510001567/1-s2.0-S0171298510001567-main.pdf?_tid=05754f9e-4f34-11e5-95a1-00000aacb360&amp;acdnat=1440952235_9b35c0a0389b3dbe95d48a4e8a583234">http://ac.els-cdn.com/S0171298510001567/1-s2.0-S0171298510001567-main.pdf?_tid=05754f9e-4f34-11e5-95a1-00000aacb360&amp;acdnat=1440952235_9b35c0a0389b3dbe95d48a4e8a583234</a>|Rui et al.: Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate</div>
+
<a class="urlextern" href="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG10823" rel="nofollow" target="_Blank" title="http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG10823">http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&amp;object=EG10823</a></div>
<div class="fn"><sup><a class="fn_bot" href="#fnt__14" id="fn__14" name="fn__14">14)</a></sup>
+
<a class="urlextern" href="http://download.springer.com/static/pdf/503/art%253A10.1007%252Fs11427-015-4887-3.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs11427-015-4887-3&amp;token2=exp=1440954389~acl=%2Fstatic%2Fpdf%2F503%2Fart%25253A10.1007%25252Fs11427-015-4887-3.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs11427-015-4887-3" rel="nofollow" target="_Blank" title="http://download.springer.com/static/pdf/503/art%253A10.1007%252Fs11427-015-4887-3.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs11427-015-4887-3&amp;token2=exp=1440954389~acl=%2Fstatic%2Fpdf%2F503%2Fart%25253A10.1007%25252Fs11427-015-4887-3.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs11427-015-4887-3">http://download.springer.com/static/pdf/503/art%253A10.1007%252Fs11427-015-4887-3.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Farticle%2F10.1007%2Fs11427-015-4887-3&amp;token2=exp=1440954389~acl=%2Fstatic%2Fpdf%2F503%2Fart%25253A10.1007%25252Fs11427-015-4887-3.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Farticle%252F10.1007%252Fs11427-015-4887-3</a>*~hmac=d600b391543effa4176063cb1502a9132af41b358eca0ec239aeb360e00506dc|Xia et al.: Insights into the function of tegument proteins from the varicella zoster virus</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__15" id="fn__15" name="fn__15">15)</a></sup>
+
<a class="urlextern" href="http://ac.els-cdn.com/S0166093411001522/1-s2.0-S0166093411001522-main.pdf?_tid=36c4b760-4f39-11e5-8e17-00000aab0f27&amp;acdnat=1440954465_433af8b92c0b2abce237c76c592225d4" rel="nofollow" target="_Blank" title="http://ac.els-cdn.com/S0166093411001522/1-s2.0-S0166093411001522-main.pdf?_tid=36c4b760-4f39-11e5-8e17-00000aab0f27&amp;acdnat=1440954465_433af8b92c0b2abce237c76c592225d4">http://ac.els-cdn.com/S0166093411001522/1-s2.0-S0166093411001522-main.pdf?_tid=36c4b760-4f39-11e5-8e17-00000aab0f27&amp;acdnat=1440954465_433af8b92c0b2abce237c76c592225d4</a>|Bäckström et al.: Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__16" id="fn__16" name="fn__16">16)</a></sup>
+
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113243/" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113243/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC113243/</a>|Mo et al.: Glycoprotein E of Varicella-Zoster Virus Enhances Cell-Cell Contact in Polarized Epithelial Cells</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__17" id="fn__17" name="fn__17">17)</a></sup>
+
<a class="urlextern" href="http://www.uptodate.com/contents/epidemiology-of-varicella-zoster-virus-infection-chickenpox" rel="nofollow" target="_Blank" title="http://www.uptodate.com/contents/epidemiology-of-varicella-zoster-virus-infection-chickenpox">http://www.uptodate.com/contents/epidemiology-of-varicella-zoster-virus-infection-chickenpox</a>|Albrecht et al.: Epidemiology of varicella-zoster virus infection: Chickenpox</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__18" id="fn__18" name="fn__18">18)</a></sup>
+
<a class="urlextern" href="http://ac.els-cdn.com/S0163445315001991/1-s2.0-S0163445315001991-main.pdf?_tid=8363e3e6-4f4f-11e5-a749-00000aab0f26&amp;acdnat=1440964043_a5145587cfd5767097caf36b486927d5" rel="nofollow" target="_Blank" title="http://ac.els-cdn.com/S0163445315001991/1-s2.0-S0163445315001991-main.pdf?_tid=8363e3e6-4f4f-11e5-a749-00000aab0f26&amp;acdnat=1440964043_a5145587cfd5767097caf36b486927d5">http://ac.els-cdn.com/S0163445315001991/1-s2.0-S0163445315001991-main.pdf?_tid=8363e3e6-4f4f-11e5-a749-00000aab0f26&amp;acdnat=1440964043_a5145587cfd5767097caf36b486927d5</a>|Grahn, Studahl: Varicella-zoster virus infections of the central nervous system -  Prognosis, diagnostics and treatment</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__19" id="fn__19" name="fn__19">19)</a></sup>
+
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811230/" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811230/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811230/</a>|Gershon: Pathogenesis and Current Approaches to Control of Varicella-Zoster Virus Infections</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__20" id="fn__20" name="fn__20">20)</a></sup>
+
<a class="urlextern" href="http://journals.cambridge.org/download.php?file=%2FHYG%2FHYG143_08%2FS0950268814002830a.pdf&amp;code=44e45de9eb264d22454f2789ffa4ab4d" rel="nofollow" target="_Blank" title="http://journals.cambridge.org/download.php?file=%2FHYG%2FHYG143_08%2FS0950268814002830a.pdf&amp;code=44e45de9eb264d22454f2789ffa4ab4d">http://journals.cambridge.org/download.php?file=%2FHYG%2FHYG143_08%2FS0950268814002830a.pdf&amp;code=44e45de9eb264d22454f2789ffa4ab4d</a>|Stamm: Syphilis: antibiotic treatment and resistance</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__21" id="fn__21" name="fn__21">21)</a></sup>
+
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807206/pdf/zcd1563.pdf" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807206/pdf/zcd1563.pdf">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807206/pdf/zcd1563.pdf</a>|Wu et al.: Evaluation of the Recombinant Protein TpF1 of <em>Treponema pallidum</em> for Serodiagnosis of Syphilis</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__22" id="fn__22" name="fn__22">22)</a></sup>
+
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225993/" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225993/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225993/</a>|Ho, Lukehart: Syphilis: using modern approaches to understand an old disease</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__23" id="fn__23" name="fn__23">23)</a></sup>
+
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360276/" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360276/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360276/</a>|Lukehart et al.: Biological Basis for Syphilis</div>
+
<div class="fn"><sup><a class="fn_bot" href="#fnt__24" id="fn__24" name="fn__24">24)</a></sup>
+
<a class="urlextern" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956094/" rel="nofollow" target="_Blank" title="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956094/">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3956094/</a>|Tampa et al.: Brief History of Syphilis</div>
+
 
</div>
 
</div>
 
</div>
 
</div>
  
 
</html>
 
</html>
<!-- Labjournal content ends here -->
 
 
{{Freiburg/wiki_content_end}}
 
{{Freiburg/wiki_content_end}}

Latest revision as of 01:16, 19 September 2015

""

E.coli genotypes

We are using E.coli strains for overexpression of our target proteins. E. coli is suitable for expression of a wide range of heterologous proteins. Our aim was the enrichment of active, soluble recombinant protein. Therefore, we used different E.coli strains and adapted different expression temperatures, durations and IPTG concentrations to get as much soluble target protein as possible. Of course, many challenges can arise during expression of recombinant proteins. That is why most expression strains have a few mutations in common to obtain a high yield of expressed proteins. Expression strains often harbor mutations that make them deficient in outer membrane protease VII and delete the Ion protease. Both of this reduces proteolysis of the expressed recombinant proteins. Additionally the native restriction/methylation system is often inactivated 1)2). Below we introduce the bacterial strains we used for protein overexpression:

Bl21 (DE3) pLysS

BL21 (DE3) is the basic IPTG-inducible strain containing T7 RNA-polymerase (DE3) for general protein expression 3). The E.coli expression strain BL21 (DE3) pLysS is useful for the expression of toxic proteins. The pLysS plasmid contains a chloramphenicol resistance. The pLysS plasmid also encodes T7 phage lysozyme, an inhibitor for T7 polymerase. In the absence of IPTG induction this inhibitor reduces and almost eliminates expression from a transformed T7 promoter containing plasmids . This inducable expression is suitable to stabilize recombinants encoding particularly toxic proteins 4)5).

Rosetta(DE3)pLysS

Rosetta™ host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid. The Rosetta strains provide a better translation for eukaryotic proteins that have not been codon optimized. In Rosetta(DE3)pLysS, the rare tRNA genes are present on the same plasmid that carries the T7 lysozyme gene for inducable expression 6).

C43 (DE3)

This overexpression strain derives from the C41 (DE3) E.coli strain. These strains contain an uncharacterized genetic mutation that allows a higher tolerance to toxic proteins. The mutation prevents E.coli cell death often associated with expression of many recombinant toxic proteins.

The advantage of this strain to BL21 (DE3) is that even membrane proteins, often particularly problematic for protein expression, can be overexpressed 7).

Received from the Toolbox - BIOSS (Centre for Biological Signalling Studies).

ArcticExpress (DE3)RP

ArcticExpress Competent Cells are engineered to address the common bacterial gene expression hurdle of protein insolubility. These cells are derived from the high-performance Stratagene BL21-Gold competent cells, enabling efficient high-level expression of heterologous proteins in E. coli. With these cells we were able to express our target proteins o/n at 10°C. Low-temperature cultivation represents one strategy for increasing the recovery of soluble protein. This is due to the fact that E. coli chaperonins, which facilitate proper protein folding by binding and stabilizing unfolded or partially folded proteins, lose activity at reduced temperatures 8)9)10).

Received from AG Weber - BIOSS (Centre of Biological Signalling Studies).

Top10

The Top10 cloning strain harbors some mutations especially useful for high efficiency cloning purposes and for propagation of plasmids. In this strain, the gene for the type I endonuclease complex KI that recognizes unmethylated DNA is not functional 11). So genes from PCR amplification are not degraded by this defense-system and can be efficiently transformed into the bacteria.
Additionally, a mutation in a nonspecific endonuclease (endA) yields cleaner preparations due to less unspecifically cut double-strand DNA 12).
Finally the rate of homologous recombination that can hinder efficient cloning is reduced by a mutation in the DNA strand exchange and recombination protein recA13).