Difference between revisions of "Part:BBa K1897001"
(→‎Construction of the composite part)
Line 14: Line 14:
 
BBa_K1897000 was first [https://parts.igem.org/Part:BBa_K1897000 created] and the other two components, BBa_K525998 and BBa_B0015 were obtained from transformation of the stock plasmids from the relevant iGEM distribution kits and subsequent PCR of the desired fragments. They were then added to BBa_K1897000 via sequential restriction enzyme digestion and ligation, creating scar sites between the individual parts and using colony PCR to check the success of the ligation reactions. The final construct was also determined by colony PCR. Colonies containing the insert of the desired size were overnight cultured in liquid culture with the relevant antibiotics, miniprepped and confirmation of the presence of the insert was done by restriction digestion to drop out the insert and PCR using pSB1C3 backbone primers VF2 and VR (Figure 1). The plasmid was also sent for sequencing to confirm that the sequence has no mutations.
 
BBa_K1897000 was first [https://parts.igem.org/Part:BBa_K1897000 created] and the other two components, BBa_K525998 and BBa_B0015 were obtained from transformation of the stock plasmids from the relevant iGEM distribution kits and subsequent PCR of the desired fragments. They were then added to BBa_K1897000 via sequential restriction enzyme digestion and ligation, creating scar sites between the individual parts and using colony PCR to check the success of the ligation reactions. The final construct was also determined by colony PCR. Colonies containing the insert of the desired size were overnight cultured in liquid culture with the relevant antibiotics, miniprepped and confirmation of the presence of the insert was done by restriction digestion to drop out the insert and PCR using pSB1C3 backbone primers VF2 and VR (Figure 1). The plasmid was also sent for sequencing to confirm that the sequence has no mutations.
  
−
[[File:HasA_complete_part_confirmation.png|300px|thumb|centre|Figure 1: DNA gel photo of confirmation of complete HasA expression in pSB1C3. Lane 1 shows the products of the restriction digestion done on the pSB1C3 plasmid containing the hasA gene using PstI and XbaI. Two bands, one approximately 2 kbp (band a) and another at around 600 bp (band b) were obtained. Lane 2 shows the product of PCR of the plasmid using pSB1C3 backbone primers VF2 and VR which shows one band at approximately 900 bp.
+
[[File:HasA_complete_part_confirmation.png|300px|thumb|centre|Figure 1: DNA gel photo of confirmation of HasA hemophore expression unit in pSB1C3. Lane 1 shows the products of the restriction digestion using PstI and XbaI. Two bands, one approximately 2 kbp (band a) and another at around 800 bp (band b) were obtained. Lane 2 shows the product of PCR of the plasmid using pSB1C3 backbone primers VF2 and VR which shows one band at approximately 1 kbp.]]
 +
 
 +
 
 +
To confirm the presence of the insert, restriction digest was done on the recombinant plasmid (lane 1) and two bands were generated. Band a represents the linearised plasmid backbone at approximately 2kbp and band b represents the insert band which has a theoretical size of 788 bp which corresponds to the size seen in the gel. PCR using the pSB1C3 primers VF2 and VR was also done (lane 2) and one clean band (band c) of approximately 1 kbp was obtained which confirms the presence of the insert as the expected size of the PCR fragment is 1074 bp.

Revision as of 17:04, 9 October 2016


HasA hemophore expression unit

Usage and Biology

The HasA hemophore (19 kDa monomer) is originally from the Serratia marcescens and used in their heme uptake pathway. When bound to heme, the HasA hemophore is able to trigger expression of genes through the Has system. The heme-bound HasA hemophore is able to bind to a membrane receptor HasR, a component of the signalling cascade that regulates expression of the genes in the has operon via HasS and HasI. When HasR is activated, it causes a conformation change in HasS, an anti-sigma factor. This causes the release of the sigma factor, HasI, initially bound to it. HasI then goes on to bind to a specific promoter to trigger expression of genes under the control of that promoter. This is a composite part created from the coding sequence of HasA (BBa K1897000) with the addition of Bba_K525998, which contains a promoter and Ribosome Binding Site (RBS), and Bba_B0015, which contains a double terminator. The addition of these parts make BBa K1897001 a functional unit that can be used for expression, unlike BBa K1897000 which lacks the promoter, RBS and terminator and hence cannot be used on its own for expression.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 480

Construction of the composite part

BBa_K1897000 was first created and the other two components, BBa_K525998 and BBa_B0015 were obtained from transformation of the stock plasmids from the relevant iGEM distribution kits and subsequent PCR of the desired fragments. They were then added to BBa_K1897000 via sequential restriction enzyme digestion and ligation, creating scar sites between the individual parts and using colony PCR to check the success of the ligation reactions. The final construct was also determined by colony PCR. Colonies containing the insert of the desired size were overnight cultured in liquid culture with the relevant antibiotics, miniprepped and confirmation of the presence of the insert was done by restriction digestion to drop out the insert and PCR using pSB1C3 backbone primers VF2 and VR (Figure 1). The plasmid was also sent for sequencing to confirm that the sequence has no mutations.

Figure 1: DNA gel photo of confirmation of HasA hemophore expression unit in pSB1C3. Lane 1 shows the products of the restriction digestion using PstI and XbaI. Two bands, one approximately 2 kbp (band a) and another at around 800 bp (band b) were obtained. Lane 2 shows the product of PCR of the plasmid using pSB1C3 backbone primers VF2 and VR which shows one band at approximately 1 kbp.


To confirm the presence of the insert, restriction digest was done on the recombinant plasmid (lane 1) and two bands were generated. Band a represents the linearised plasmid backbone at approximately 2kbp and band b represents the insert band which has a theoretical size of 788 bp which corresponds to the size seen in the gel. PCR using the pSB1C3 primers VF2 and VR was also done (lane 2) and one clean band (band c) of approximately 1 kbp was obtained which confirms the presence of the insert as the expected size of the PCR fragment is 1074 bp.