Difference between revisions of "Part:BBa K1897000"

(→‎Usage and Biology)
(→‎Transformation and characterisation of the HasA gene)
 
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The sequence of HasA was obtained and modifications were done to the original sequence for the addition of a hexa histidine tag in the reading frame. This sequence was then sent for synthesis by IDT Technologies. The HasA gene obtained however is in the company vector and therefore had to be removed and placed into the pSB1C3 by restriction digestion and ligation. The ligation mix was then transformed into E. coli DH5α and colonies were then screened via colony PCR. Colonies containing the insert of the desired size were overnight cultured in liquid culture, miniprepped and confirmation of the presence of the insert was done by restriction digestion to drop out the insert as well as PCR of using pSB1C3 backbone primers VF2 and VR (Figure 1). The plasmid was also sent for sequencing to confirm that the sequence has no mutations.  
 
The sequence of HasA was obtained and modifications were done to the original sequence for the addition of a hexa histidine tag in the reading frame. This sequence was then sent for synthesis by IDT Technologies. The HasA gene obtained however is in the company vector and therefore had to be removed and placed into the pSB1C3 by restriction digestion and ligation. The ligation mix was then transformed into E. coli DH5α and colonies were then screened via colony PCR. Colonies containing the insert of the desired size were overnight cultured in liquid culture, miniprepped and confirmation of the presence of the insert was done by restriction digestion to drop out the insert as well as PCR of 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_only_bb_part_confirmation.png|300px|thumb|centre|Figure 1: DNA gel photo of confirmation of HasA gene 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_only_bb_part_confirmation_negative.png|300px|thumb|centre|Figure 1: DNA gel photo of confirmation of HasA gene 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.]]
  
 
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 611 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 900 bp was obtained which confirms the presence of the insert as the expected size of the PCR fragment is 899 bp.
 
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 611 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 900 bp was obtained which confirms the presence of the insert as the expected size of the PCR fragment is 899 bp.

Latest revision as of 21:38, 10 October 2016


HasA hemophore coding sequence

HasA hemophore coding sequence


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 442

Usage and Biology

The HasA hemophore is a monomer of 19 kDa originally used in the Serratia marcescens 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 antisigma factor. This causes the release of the sigma factor initially bound to it, HasI. HasI then triggers expression of genes under the control of the Has promoter.

Transformation and characterisation of the HasA gene

The sequence of HasA was obtained and modifications were done to the original sequence for the addition of a hexa histidine tag in the reading frame. This sequence was then sent for synthesis by IDT Technologies. The HasA gene obtained however is in the company vector and therefore had to be removed and placed into the pSB1C3 by restriction digestion and ligation. The ligation mix was then transformed into E. coli DH5α and colonies were then screened via colony PCR. Colonies containing the insert of the desired size were overnight cultured in liquid culture, miniprepped and confirmation of the presence of the insert was done by restriction digestion to drop out the insert as well as PCR of 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 gene 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.

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 611 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 900 bp was obtained which confirms the presence of the insert as the expected size of the PCR fragment is 899 bp.