Difference between revisions of "Part:BBa K3788004:Design"
Line 8: Line 8:
 
===Design Notes===
 
===Design Notes===
 
<p>After the sequence eventual mutation was prevented : Amino acids have been changed to avoid the formation of a stop codon and also to remove restriction sites corresponding to RFC10 and RFC25.</p>
 
<p>After the sequence eventual mutation was prevented : Amino acids have been changed to avoid the formation of a stop codon and also to remove restriction sites corresponding to RFC10 and RFC25.</p>
 +
 +
 +
<p> <u> Creating the BBa_K3788004 part </u> </p>
 +
 +
<p>As for the cloning of the part BBa_K3788004 into the plasmid pBAD24 MSC3, the following strategy was thought. The plasmid pBAD24_6his<i>p20</i>_flag<i>cry11Aa</i> (BBa_K3788003 part into pBAD24 vector) can be used after a digested with KpnI  restriction enzyme and used to insert the gene strep<i<cyt1Aa</i> (BBa_K3788000) by recombination by the SLIC cloning method.</i>
 +
 +
<p> <i><u> Cloning strategy table </u></i>:  Thus, strepcyt1Aa (BBa_K3788000) has to be amplified by PCR with the specifics primers to be able to insert strep<i>cyt1Aa</i> gene into pBAD24_6his<i>p20</i>_flag<i>cry11Aa</i> . </p>
 +
 +
https://2021.igem.org/wiki/images/e/e8/T--Aix-Marseille--toxto-cloning-strategy.png
 +
  
  

Revision as of 16:01, 15 October 2021


6His-P20, Flag-Cry11Aa and Strep-Cyt1Aa coding sequence


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 154
    Illegal XhoI site found at 2438
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

After the sequence eventual mutation was prevented : Amino acids have been changed to avoid the formation of a stop codon and also to remove restriction sites corresponding to RFC10 and RFC25.


Creating the BBa_K3788004 part

As for the cloning of the part BBa_K3788004 into the plasmid pBAD24 MSC3, the following strategy was thought. The plasmid pBAD24_6hisp20_flagcry11Aa (BBa_K3788003 part into pBAD24 vector) can be used after a digested with KpnI restriction enzyme and used to insert the gene strep<i<cyt1Aa</i> (BBa_K3788000) by recombination by the SLIC cloning method.</i> <p> Cloning strategy table : Thus, strepcyt1Aa (BBa_K3788000) has to be amplified by PCR with the specifics primers to be able to insert strepcyt1Aa gene into pBAD24_6hisp20_flagcry11Aa .

T--Aix-Marseille--toxto-cloning-strategy.png


Source

The 6His_P20__Flag_cry11Aa__Strep_cyt1Aa sequence is made from BBa_K3788002, BBa_K3788001 (BBa_K3788003) and BBa_K3788000 parts.

Theses 2 parts are optimised sequences for E. coli obtain from Part:BBa_K2938008, Part:BBa_K2938005 and Part:BBa_K2938003.


References

- Bravo, A., Gill, S. S. & Soberón, M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49, 423–435 (2007).

- Hua, G., Masson, L., Jurat-Fuentes, J. L., Schwab, G. & Adang, M. J. Binding Analysis of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis. Appl. Environ. Microbiol. 67, 872–879 (2001).

- Manasherob, R. et al. Effect of Accessory Proteins P19 and P20 on Cytolytic Activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli. Curr. Microbiol. 43, 355–364 (2001).