Difference between revisions of "Part:BBa K1621003"

 
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<partinfo>BBa_K1621003 short</partinfo>
 
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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 Clostridium tetani. 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 C. tetani but cannot transmit the infection to other individuals.
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This part contains a coding sequence belonging to the tetanus neurotoxin. It is not responsible for the toxic properties and therefore exhibits no toxicity in humans.
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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. The latter causes damage to the heart muscle and blood components.
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After an infection with C. tetani 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.
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To avoid long-term effects due to an infection with C. tetani 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.
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The source organism of this part is '''Clostridium tetani'', an anaerobic, gram-positive bacterium causing tetanus disease. ''C. tetani'' produces two different toxins, tetanolysin and tetanospasmin. The first is responsible for damage of the heart muscle and blood components. The latter causes the more prominent symptoms like hypersensitivity, increased reactions and spasms.
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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. 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. 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.
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The neurotoxin (or tetanospasmin) is composed of a heavy and a light chain. They are translated as one polypeptide of about 150 kDa and cleaved by a protease afterwards. The resulting heavy (~100 kDa) and light chain (~50kDa) are linked to each other by two disulfide bonds (Yu ''et al.'', 2011).
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Regarding the functions, the light chain is the toxic part of the toxin that is translocated into the neuron at the neuromuscular junction. The heavy chain’s function is to mediate this translocation. Therefore, the C-terminal part interacts with the cells membrane via specific receptors and the N-terminal part aids in the translocation process itself. In the cell, the light chain prevents the exocytosis of neurotransmitters (Rummel ''et al.'', 2003).  
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Nonetheless, the C-terminal part of the heavy chain which is encoded by this part is promising in the development of second generation vaccines against the toxin itself (Yu ''et al.'', 2011). This means of course, that it provokes an immune response in humans and work has to be done under consideration of the respective safety requirements.  
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As Yu ''et al.'' (2011) showed before, cloning the sequence into an expression vector enables efficient overexpression of the part. [Results will be updated]
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The part has been shipped to the registry in standard pSB1C3 and begins with a start codon (ATG). Cloning into the chipping backbone was performed by Gibson Assembly and the sequence was verified afterwards.  
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 06:25, 30 August 2015

TeNT_Hc - C-terminal part of the heavy chain (tetanus toxin)

This part contains a coding sequence belonging to the tetanus neurotoxin. It is not responsible for the toxic properties and therefore exhibits no toxicity in humans.

The source organism of this part is 'Clostridium tetani, an anaerobic, gram-positive bacterium causing tetanus disease. C. tetani produces two different toxins, tetanolysin and tetanospasmin. The first is responsible for damage of the heart muscle and blood components. The latter causes the more prominent symptoms like hypersensitivity, increased reactions and spasms.

The neurotoxin (or tetanospasmin) is composed of a heavy and a light chain. They are translated as one polypeptide of about 150 kDa and cleaved by a protease afterwards. The resulting heavy (~100 kDa) and light chain (~50kDa) are linked to each other by two disulfide bonds (Yu et al., 2011).
Regarding the functions, the light chain is the toxic part of the toxin that is translocated into the neuron at the neuromuscular junction. The heavy chain’s function is to mediate this translocation. Therefore, the C-terminal part interacts with the cells membrane via specific receptors and the N-terminal part aids in the translocation process itself. In the cell, the light chain prevents the exocytosis of neurotransmitters (Rummel et al., 2003).

Nonetheless, the C-terminal part of the heavy chain which is encoded by this part is promising in the development of second generation vaccines against the toxin itself (Yu et al., 2011). This means of course, that it provokes an immune response in humans and work has to be done under consideration of the respective safety requirements.

As Yu et al. (2011) showed before, cloning the sequence into an expression vector enables efficient overexpression of the part. [Results will be updated]

The part has been shipped to the registry in standard pSB1C3 and begins with a start codon (ATG). Cloning into the chipping backbone was performed by Gibson Assembly and the sequence was verified afterwards.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 430
  • 1000
    COMPATIBLE WITH RFC[1000]