Difference between revisions of "Part:BBa K1897001"
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SDS-PAGE of the samples was run on a polyacrylamide gel (4% stacking, 10% resolving) at 70V for 30 minutes and followed by 100V for 1 hour. Subsequently, semi-dry transfer was conducted at 15V for 1 hour to transfer the protein bands to a PVDF membrane (Figure 2). | SDS-PAGE of the samples was run on a polyacrylamide gel (4% stacking, 10% resolving) at 70V for 30 minutes and followed by 100V for 1 hour. Subsequently, semi-dry transfer was conducted at 15V for 1 hour to transfer the protein bands to a PVDF membrane (Figure 2). | ||
| − | [[File:HasA_WB.png| | + | [[File:HasA_WB.png|300px|thumb|left|Figure 2: Western blot of his-tagged HasA expression and purification. The wells were loaded with (left to right) total lysate, flowthrough, wash 1, wash 2, elute 1, elute 2, and elute 3.]] |
Revision as of 19:14, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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.
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.
Purification, SDS-PAGE and Western blot of HasA protein
The plasmid was transformed into E. coli BL21(DE3) for expression. As it is under a T7 promoter, the induction of protein expression was done using Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the bacteria allowed to grow for 3 hours. The bacteria were then pelleted and lysed using B-PER Bacterial Protein Extraction Reagent. Centrifugation was then done and the supernatent ran through the Nickel Chelated Column (flowthrough). The column was then washed twice with wash buffer (wash 1 and wash 2) and three elutions (1mL each) were collected.
SDS-PAGE of the samples was run on a polyacrylamide gel (4% stacking, 10% resolving) at 70V for 30 minutes and followed by 100V for 1 hour. Subsequently, semi-dry transfer was conducted at 15V for 1 hour to transfer the protein bands to a PVDF membrane (Figure 2).
