Difference between revisions of "Part:BBa K3333017"
 
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HA participate in gene recombination between donor plasmid and phage genomes. Some fragments of phage DNA are selected as homologous arms, which are divided into HA-up and HA-down, and these two sequences are connected to the upstream and downstream of gene need to be inserted respectively by overlap PCR.
 
HA participate in gene recombination between donor plasmid and phage genomes. Some fragments of phage DNA are selected as homologous arms, which are divided into HA-up and HA-down, and these two sequences are connected to the upstream and downstream of gene need to be inserted respectively by overlap PCR.
  
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Different from HA-Up(orf 73) we designed before, orf 53 in phage's genome is selected as another homologous arm. Orf53 is predicted to code for the holin protein which play an important role in host lysis. Since we aim to design an non-host-lysis phage that kill the bacteria with toxin protein, orf53 is supposed to be knocked out. So, we constructed a composite part "HA-Up(orf73) - relE -tat promoter - HA-Down(orf73)".With CRISPR-Cpf1 and lambda-red system, relE, a toxin protein, and tat promoter, its constitutive promoter, will replace holin gene. In this way, holin gene could be replaced by relE-tat promoter. Click for more information about Cpf1, lambda-red system, relE and tat promoter.
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Different from HA-Up(orf 73) we designed before, orf 53 in phage's genome is selected as another homologous arm. Orf53 is predicted to code for the holin protein which play an important role in host lysis. Since we aim to design an non-host-lysis phage that kill the bacteria with toxin protein, orf53 is supposed to be knocked out. So, we constructed a composite part "HA-Up(orf53) - relE -tat promoter - HA-Down(orf53)".With CRISPR-Cpf1 and lambda-red system, relE, a toxin protein, and tat promoter, its constitutive promoter, will replace holin gene. In this way, holin gene could be replaced by relE-tat promoter. Click [https://parts.igem.org/Part:BBa_K3333015] and [https://parts.igem.org/Part:BBa_K3333011] for more information about Cpf1, lambda-red system, relE and tat promoter.
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Latest revision as of 14:24, 14 October 2020


HA-Up (orf53)

Usage and Biology

HA participate in gene recombination between donor plasmid and phage genomes. Some fragments of phage DNA are selected as homologous arms, which are divided into HA-up and HA-down, and these two sequences are connected to the upstream and downstream of gene need to be inserted respectively by overlap PCR.

Different from HA-Up(orf 73) we designed before, orf 53 in phage's genome is selected as another homologous arm. Orf53 is predicted to code for the holin protein which play an important role in host lysis. Since we aim to design an non-host-lysis phage that kill the bacteria with toxin protein, orf53 is supposed to be knocked out. So, we constructed a composite part "HA-Up(orf53) - relE -tat promoter - HA-Down(orf53)".With CRISPR-Cpf1 and lambda-red system, relE, a toxin protein, and tat promoter, its constitutive promoter, will replace holin gene. In this way, holin gene could be replaced by relE-tat promoter. Click [1] and [2] for more information about Cpf1, lambda-red system, relE and tat promoter.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 180
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 98
    Illegal SapI.rc site found at 415