Difference between revisions of "Part:BBa K2739000:Design"

Latest revision as of 01:18, 18 October 2018

ProR-PhaR (The phasin autoregulation system with native promoter)

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 253

Design Notes

This composite part is consisted PhaR and its native promoter. This part was designed to investigate its relation in PHA production through PHA operon with and without phasin. The promoter used in this part is native to phaR and R.eutropha. Sequence of PhaR and its native promoter could be found in iGEM registry, as two separate basic parts (BBa_K108015 and BBa_K108014) and being not available. The source of these sequences were recorded as 1. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology. 2002, 148:2413–2426. and 2. NCBI (with no further information), having no mentioned if the sequence was codon optimised.

We access the online NCBI data and found out the current available phaR sequence is being 4 bp different to BBa_K108015; and the functional promoter is being 134bp shorter. Therefore we have used the new phaR and promoter sequence as our new template. Due to the excessive GC in the sequence, we have it codon optimised to E.coli and generated the part through the IDT company service.

Figure 1. Comparison of phaR sequence from iGEM08_Tsinghua (BBa_K108015) and online NCBI(AM260479.1). Capital letters: NCBI(AM260479.1) and Small letter: iGEM08_Tsinghua (BBa_K108015).

Figure 2. Comparison of phaR sequence before and after codon optimisation. Capital letters: NCBI (AM260479.1) and Small letter: codon optimised version (BBa_K2739001).

Source

BBa_K108015, BBa_K108014 and NCBI (AM260479.1)

ProR and PhaR Sequence selection based on Reference 1 information.

PhaR sequence codon optimised through IDT

References

1. Yamada M, Takahashi S, Okahata Y, Doi Y, Numata K. (2013 ) Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]. AMB Express. 3(1):6.

@@ Line 14: / Line 14: @@
 https://static.igem.org/mediawiki/parts/c/cd/T--Edinburgh_OG_BBa_K2739000--image_9.jpeg
-Figure 1. Comparison of phaR sequence from  iGEM08_Tsinghua and online NCBI. Capital letters: NCBI (AM260479.1) and Small letter: iGEM08_Tsinghua.
+https://static.igem.org/mediawiki/parts/c/cd/T--Edinburgh_OG_BBa_K2739000--image_9.jpeg
+Figure 1. Comparison of phaR sequence from  iGEM08_Tsinghua (BBa_K108015) and online NCBI(AM260479.1). Capital letters: NCBI(AM260479.1)  and Small letter: iGEM08_Tsinghua (BBa_K108015).
 https://static.igem.org/mediawiki/parts/9/9a/T--Edinburgh_OG_BBa_K2739000--image_10.jpeg
-Figure 2. Comparison of phaR sequence before and after codon optimisation. Capital letters: NCBI (AM260479.1) and Small letter: codon optimised version (BBa_K2739001).
+Figure 2. Comparison of phaR sequence before and after codon optimisation. Capital letters:  NCBI (AM260479.1) and Small letter: codon optimised version (BBa_K2739001).