Difference between revisions of "Part:BBa K4604035:Design"
 
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     <caption><b>Fig. 1:</b> table showing parameters varying from PCR protocol for piG_08</caption>
 
     <caption><b>Fig. 1:</b> table showing parameters varying from PCR protocol for piG_08</caption>
 
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Since regular Golden Gate cloning did not work with the plasmid we used a different approach. We fused the single parts (see in the table above) together via PCR to one large fragment. Therefore we designed complementary overhangs attached to the primers used to amplify the single fragments. First we fused the amp promoter with <i>mazE</i> and then added the terminator. This insert was amplified according to the Q5 protocol with 62°C annealing temperature and an elongation time of 1 minute. The backbone pGGAselect and this PCR fragment were then digested with PacI and NotI for approximately 30 minutes. Afterwards the fragments were purified from gel. A ligation was set up according to the protocol, afterwards a transformation was done. The resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from  overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.  
 
Since regular Golden Gate cloning did not work with the plasmid we used a different approach. We fused the single parts (see in the table above) together via PCR to one large fragment. Therefore we designed complementary overhangs attached to the primers used to amplify the single fragments. First we fused the amp promoter with <i>mazE</i> and then added the terminator. This insert was amplified according to the Q5 protocol with 62°C annealing temperature and an elongation time of 1 minute. The backbone pGGAselect and this PCR fragment were then digested with PacI and NotI for approximately 30 minutes. Afterwards the fragments were purified from gel. A ligation was set up according to the protocol, afterwards a transformation was done. The resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from  overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.  
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===Source===
 
===Source===
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The Amp promoter was taken by iGEM ECUST 2017 (BBa_K2308010). Assembled via a PCR annealing technique and Gibson Assembly.
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The Amp promoter was taken by iGEM ECUST 2017 (<a href="https://parts.igem.org/Part:BBa_K2308010">BBa_K2308010</a>).</html>
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===References===
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Latest revision as of 01:02, 12 October 2023


piG_08 (ampProm_mazE)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 226
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 515


Design Notes

Choice of a constitutive promoter to ensure baseline expression of MazE (antitoxin).

Cloning of piG_08

The amp promoter as well as the rrnB terminator were used for cloning from a plasmid of iGEM2022 Freiburg team. The mazE was synthesized by IDT, the sequence for that was taken from the Library of Medicine (gene ID:947245) out of E. coli K12 MG1655 . For the PCR we used the general protocol for the Q5 polymerase with varying parameters for elongation time and annealing temperature:

Fig. 1: table showing parameters varying from PCR protocol for piG_08
fragment Annealing temp. Elongation time Fragment size (in bp)
Amp promoter 61°C 15 s 100
mazE 61°C 20 s 320
rrnB terminator 61°C 30 s 450


Since regular Golden Gate cloning did not work with the plasmid we used a different approach. We fused the single parts (see in the table above) together via PCR to one large fragment. Therefore we designed complementary overhangs attached to the primers used to amplify the single fragments. First we fused the amp promoter with mazE and then added the terminator. This insert was amplified according to the Q5 protocol with 62°C annealing temperature and an elongation time of 1 minute. The backbone pGGAselect and this PCR fragment were then digested with PacI and NotI for approximately 30 minutes. Afterwards the fragments were purified from gel. A ligation was set up according to the protocol, afterwards a transformation was done. The resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing for correct insertion and no mutation.




Source

The Amp promoter was taken by iGEM ECUST 2017 (BBa_K2308010).