Difference between revisions of "Part:BBa K1897007:Design"

Revision as of 20:11, 11 October 2016

Complete Has operon (controlling expression of luxR)

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1691
Illegal NheI site found at 1714
Illegal NotI site found at 3609
Illegal NotI site found at 4403
Illegal NotI site found at 4527
Illegal NotI site found at 5531
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1720
Illegal BamHI site found at 4476
Illegal BamHI site found at 4810
Illegal BamHI site found at 5466
Illegal XhoI site found at 1
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1767
Illegal NgoMIV site found at 1771
Illegal NgoMIV site found at 1827
Illegal NgoMIV site found at 1945
Illegal NgoMIV site found at 2099
Illegal NgoMIV site found at 2164
Illegal NgoMIV site found at 2500
Illegal AgeI site found at 1404
Illegal AgeI site found at 1516
1000
COMPATIBLE WITH RFC[1000]

Design Notes

BBa_1897007 is a complex part that has many components. For the Has operon genes HasR, HasS and HasI, there is a hexa histidine tag added before the end of the coding sequence to allow for easy protein purification and identification on a Western Blot. The tag's position at the C terminus is especially important for HasR where it has been shown that the N terminal of HasR is especially crucial for signal transduction (Biville et al., 2004). A mRFP gene was included after the luxR (the target gene to be expressed for the RIOTSystem under NUS_Singapore) as a visual indicator to show the induction of the circuit in the presence of holo-HasA. To determine if the Has proteins under the constitutive promoter are expressed, an Ampicillin resistance gene (BBa_P1002) was included after the HasI gene. Thus, only E. coli that have successful expression of the Has operon in pSB1C3 can grow on double antibiotic plates containing Ampicillin and Chlorophenicol.

Furthermore, there are enzyme restriction sites that flank the Has operon genes to allow selective removal of any of the genes from the complete operon. This can be used to validate the role of each signalling protein in the signalling cascade by selective removal of different Has proteins.

Source

The complete Has operon signalling systme consists of many genes that have been synthesised together. The sequences for the original Has system genes that encode the signalling proteins (HasR, HasS and HasI) were obtained from the Serratia marcescens genome and documented in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2). The Has operon promoter was discovered and discussed by Biville et al. (2004) and we obtained the sequence based on their results.The luxR gene (BBa_C0062), Ribosome Binding Site (BBa_B0034), mRFP (BBa_E1010), terminators (BBa_B0015) and Ampicillin resistance gene (BBa_P1002) are all Biobrick parts and their sequences obtained from the Registry of Standard Biological Parts. There are also

References

Biville, F., Cwerman, H., Létoffé, S., Rossi, M. S., Drouet, V., Ghigo, J. M., & Wandersman, C. (2004). Haemophore‐mediated signalling in Serratia marcescens: a new mode of regulation for an extra cytoplasmic function (ECF) sigma factor involved in haem acquisition. Molecular microbiology, 53(4), 1267-1277.

Cescau, S., Cwerman, H., Letoffe, S., Delepelaire, P., Wandersman, C., & Biville, F. (2007). Heme acquisition by hemophores. Biometals, 20(3-4), 603-613.

Rossi, M. S., Paquelin, A., Ghigo, J. M., & Wandersman, C. (2003). Haemophore‐mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules. Molecular microbiology, 48(6), 1467-1480.

Wandersman, C., & Delepelaire, P. (2004). Bacterial iron sources: from siderophores to hemophores. Annu. Rev. Microbiol., 58, 611-647.

@@ Line 7: / Line 7: @@
 ===Design Notes===
-NIL
+BBa_1897007 is a complex part that has many components. For the Has operon genes HasR, HasS and HasI, there is a hexa histidine tag added before the end of the coding sequence to allow for easy protein purification and identification on a Western Blot. The tag's position at the C terminus is especially important for HasR where it has been shown that the N terminal of HasR is especially crucial for signal transduction (Biville ''et al.'', 2004). A mRFP gene was included after the luxR (the target gene to be expressed for the RIOTSystem under NUS_Singapore) as a visual indicator to show the induction of the circuit in the presence of holo-HasA. To determine if the Has proteins under the constitutive promoter are expressed, an Ampicillin resistance gene (BBa_P1002) was included after the HasI gene. Thus, only ''E. coli'' that have successful expression of the Has operon in pSB1C3 can grow on double antibiotic plates containing Ampicillin and Chlorophenicol.
+Furthermore, there are enzyme restriction sites that flank the Has operon genes to allow selective removal of any of the genes from the complete operon. This can be used to validate the role of each signalling protein in the signalling cascade by selective removal of different Has proteins.
 ===Source===