Difference between revisions of "Part:BBa K1897001"

(→‎Purification, SDS-PAGE and Western blot of HasA protein)
 
(One intermediate revision by the same user not shown)
Line 14: Line 14:
 
[[File:HasA expression unit diagram.png|400px|centre|thumb|Figure 1: Schematic of the complete HasA expression unit.]]
 
[[File:HasA expression unit diagram.png|400px|centre|thumb|Figure 1: Schematic of the complete HasA expression unit.]]
  
−
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 2). The plasmid was also sent for sequencing to confirm that the sequence has no mutations.
  
 
[[File:HasA_complete_part_confirmation_negative.png|300px|thumb|centre|Figure 2: 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.]]
 
[[File:HasA_complete_part_confirmation_negative.png|300px|thumb|centre|Figure 2: 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.]]
Line 30: Line 30:
 
As seen in figure 3, intense bands were observed in the total lysate and elute fractions, with the thickest band in the elute 2. The presence of HasA in the total lysate could be due to the saturation of the column and therefore some protein was lost when the total lysate was initially put through the column. A band at the approximate size of 20 kDa is found in all the elute fractions with the thickest, and hence the greatest concentration of HasA, in the elute fraction 2.
 
As seen in figure 3, intense bands were observed in the total lysate and elute fractions, with the thickest band in the elute 2. The presence of HasA in the total lysate could be due to the saturation of the column and therefore some protein was lost when the total lysate was initially put through the column. A band at the approximate size of 20 kDa is found in all the elute fractions with the thickest, and hence the greatest concentration of HasA, in the elute fraction 2.
  
−
===Induction of Has system===
+
<!--===Induction of Has system===
  
 
The HasA was then used to induce the Has system circuit ([https://parts.igem.org/Part:BBa_K1897007 BBa_K1897007]) that has been transformed into ''E. coli''. The circuit contains a RFP to indicate that the has operon has been successfully expressed. The bacteria were exposed to different concentrations of holo-HasA and the bacteria were photographed using fluorescence microscopy.
 
The HasA was then used to induce the Has system circuit ([https://parts.igem.org/Part:BBa_K1897007 BBa_K1897007]) that has been transformed into ''E. coli''. The circuit contains a RFP to indicate that the has operon has been successfully expressed. The bacteria were exposed to different concentrations of holo-HasA and the bacteria were photographed using fluorescence microscopy.
  
−
<!--[[File:Induction of has operon.png|800px|thumb|centre|Figure 3: HasA induction of ''E. coli'' with the Has System circuit. Top: red fluorescence microscopy pictures of (from left to right) negative control, 10<sup>-4</sup> M HasA, 10<sup>-5</sup> M HasA. Bottom: bright field microscopy pictures of (from left to right) negative control, 10<sup>-4</sup> M HasA, 10<sup>-5</sup> M HasA]]
+
[[File:Induction of has operon.png|800px|thumb|centre|Figure 3: HasA induction of ''E. coli'' with the Has System circuit. Top: red fluorescence microscopy pictures of (from left to right) negative control, 10<sup>-4</sup> M HasA, 10<sup>-5</sup> M HasA. Bottom: bright field microscopy pictures of (from left to right) negative control, 10<sup>-4</sup> M HasA, 10<sup>-5</sup> M HasA]]
  
 
As seen in figure 3, in the absence of holo-HasA, the circuit is not activated hence the ''E. coli'' do not fluoresce under excitation. However, upon the addition of holo-HasA, the bacteria are observed to be red, which indicates the successful activation of the Has system and hence the expression of the RFP.-->
 
As seen in figure 3, in the absence of holo-HasA, the circuit is not activated hence the ''E. coli'' do not fluoresce under excitation. However, upon the addition of holo-HasA, the bacteria are observed to be red, which indicates the successful activation of the Has system and hence the expression of the RFP.-->

Latest revision as of 22:15, 18 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 trigger expression of genes under the control of that promoter. This is a composite part created from the coding sequence of HasA 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

Figure 1: Schematic of the complete HasA expression unit.

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 2). The plasmid was also sent for sequencing to confirm that the sequence has no mutations.

Figure 2: 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.

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. After blocking and incubation with the relevant primary and secondary antibodies, the blot was subjected to a homemade substrate and the bands were visualised with chemiluminescence (Figure 2).

Figure 3: 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.

As seen in figure 3, intense bands were observed in the total lysate and elute fractions, with the thickest band in the elute 2. The presence of HasA in the total lysate could be due to the saturation of the column and therefore some protein was lost when the total lysate was initially put through the column. A band at the approximate size of 20 kDa is found in all the elute fractions with the thickest, and hence the greatest concentration of HasA, in the elute fraction 2.