Coding

Part:BBa_K2074023

Designed by: Yi Cai   Group: iGEM16_FAFU-CHINA   (2016-10-09)


Cry11Aa(Codon optimization)-Extended FMDV

There is a overview about parts which 2016 FAFU-CHINA created.

800px-T--FAFU-CHINA--partsclones.png

When we made the draft plan about our project, we noticed that NCTU_Formosa 2010’s project made use of Cry11Aa gene. And they constructed the related part BBa_K332011. In their project, they transformed the Cry11Aa gene into E. coli to kill the larvae of mosquitoes. Considering the potential safety problem, using E. coli in the natural environment is not a good idea. Therefore, we decided to use Chlamydomonas reinhardti to express toxin genes such as Cry11Aa. Firstly, we wanted to ask them to send this part to us. And we can transform it to Chlamydomonas reinhardti to test our idea. But we noticed that the codon bias of Chlamydomonas reinhardti was significantly different from that of E. coli. At last, we decided to synthesize the codon-optimized DNA sequence to replace it. We have mentioned in the wiki that we co-expressed Cry and Cyt by 2A-peptide system. So we submit the codon-optimized Cry11Aa with 2A peptide part BBa_K2074023. Users can link the other genes by infusion technology. Based on the result of SWISS-Model, 2A peptide residues will mot affect the function of toxins. If you are interested in the details, you can visit links as following: http://parts.igem.org/wiki/index.php?title=Part:BBa_K332011


Cry11Aa is cloned from Bacillus thuringiensis. It shows specific toxicity to Culex and Aedes by bioassay. The Cry protein is consisted of three functional domains. Domain I is a seven α-helices bundle. It can insert itself into a membrane by using its hydrophobic helices α4 and α5 to insert into the phospholipid bilayer. The pore formation occurs on its α3 helix. Domain II and domain III are two β-sheets which are involved in the receptor interactions. Domain II contains extremely variable loops, which are the binding site of the receptor. Domain III has the function of stabilizing the toxin. Cyt proteins have a single α-β domain which do not bind to receptors but can directly insert into the cell membrane and then form a pore causing cell death. Although Cry and Cyt proteins are two big families of δ-endotoxins, they are far related.Cyt1 and Cyt2 are two types of Cyt proteins found in Bti.. Generally, Cry proteins are believed to exert toxicity by interacting with the proteins on the brush border membrane and then insert into the membrane which takes multiple steps. At the beginning in mosquitoes' gut, the crystalline inclusions are cleaved at the disulfide bond to release the Cry pre-toxin. Then the soluble proteins are activated by being cleaved again by intestinal protease. When toxins reach to the brush border membrane microvilli, they bind to the proteins, or known as receptors on the membrane. The binding process takes two step. Firstly, the monomeric Cry toxin binds to cadherin, resulting in the formation of pre-pore oligomer as Figure1.

Figure.1 The Mechanism of Cry and Cyt Toxins

Then the oligomer binds to a GPI-anchored APN or ALP. Secondly, the previous binding induces the oligomer insertion into the lipid rafts membrane. A formation of ion permeable pore is followed by the insertion which allows small molecules to pass through the membrane. The membrane potential inevitably changes greatly, causing the swelling of cell and finally breaking down. When the cell lysis reaches to a certain degree, the midgut necrosis and epithelial denaturation follow. Then, the alkaline hypertonic inclusions in midgut enters into hemocoel and the pH of haemolymph rises causing paralysis of larvae and finally death. To increase the express level in Chlamydomonas reintmrdtii, we synthesis Chlamydomonas reintmrdtii codon optimized Cry11Aa. Meanwhile, we added 2A peptide sequence at the end of 5’ According to the published paper and the result of Swiss-model, there is no effect to the toxicity of Cry or Cyt. Users can use infusion technology to link the express vector to express toxins. This part is the improvement of BBa_K332011 based on Chlamydomonas reintmrdtii codon optimization.

K2074023.png

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 517
    Illegal NgoMIV site found at 1681
    Illegal NgoMIV site found at 2029
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 49
    Illegal BsaI.rc site found at 244
    Illegal BsaI.rc site found at 1459
    Illegal SapI.rc site found at 1440


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