Difference between revisions of "Part:BBa K4604027:Design"
 
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===Cloning of piG_K12BSb===
 
===Cloning of piG_K12BSb===
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The lacI promoter, <i>lacI</i> gene, trc promoter, sfGFP and rrnB terminator were given to us from iGEM Freiburg 2022 on a plasmid called pIG23_04. We took plasmid pIG23_03 (also given to us from iGEM Freiburg 2022) as our backbone, including an antibiotic resistance (spectinomycin) gene and Ori. SfGFP, trc promoter, <i>lacI</i> and lacI promoter were taken from pIG23_04 and cloned into pIG23_03 using Gibson Assembly and AQUA cloning. Both plasmids were templates for PCR, to amplify the parts and add the correct overhangs via gibson primers.
 
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After assembling the two plasmid parts, this new plasmid, the sensor backbone, was again used as PCR template to clone the correct overhangs for the insertion of the riboswitch between lacI promoter and <i>lacI</i> gene.
 
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The sequence for riboswitch K12 originates from <i>E. coli K12</i> from the 5´-UTR of the <i>btuB</i> gene. We ordered the DNA for the K12 riboswitch from IDT and used it as a template. For the PCR we used the general protocol for the Q5 polymerase with varying parameters (elongation time and annealing temperature):
 
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<table border=3 style="margin-left:auto;margin-right:auto;">
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Plasmid piG_K12BSa (<a href="https://parts.igem.org/Part:BBa_K4604026">BBa_K4604026</a>) functioned as a template for a PCR to insert a stronger RBS from the TetR promoter (modified T7 RBS) directly behind the riboswitch. Gibson primer were designed to have the RBS as their overhang/complementary region. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 72°C and an elongation time of 6 minutes. A Dpn1 digest was done at 37°C overnight, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Gibson Assembly and AQUA cloning was used according to the protocols to assemble the plasmid. A transformation was done. As the change in the plasmid is only a RBS with a length of merely 14 bp a colony PCR was not possible to perform as screening methode, because the difference in length wouldn't have been noticeable on the gel. Instead DNA of several colonies was isolated from overnight cultures (5mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to check for correct insertion and no mutation.</html>
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<caption><b>Fig. 1:</b> table showing parameters varying from PCR protocol for piG_K12BSb</caption>
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    <th>fragment</th>
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    <th>Annealing temp.</th>
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    <th>Elongation time</th>  
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    <th>Fragment size (in bp)</th>
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  </tr>
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  <tr>
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    <th>trc promoter, <i>sfGFP</i>, lacI promoter, <i>lacI</i> gene</th>
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    <th>72°C</th>
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    <th>1.5 min</th>
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    <th>2670</th>
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  </tr>
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  <tr>
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    <th>backbone (Ori and resistance)</th>
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    <th>72°C</th>
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    <th>1.5 min</th>
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    <th>2080</th>
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</tr>
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<tr>
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    <th>K12 riboswitch</th>
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    <th>65°C</th>
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    <th>30 s</th>
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    <th>250</th>
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</tr>
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<tr>
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    <th>create overhangs to add riboswitch between lacI prom. and <i>lacI</i></th>
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    <th>62°C</th>
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    <th>6 min</th>
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    <th>4700</th>
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</tr>
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</table></figure>
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</html>
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The DNA of the plasmid fragments, pIG23_04 and pIG23_03, was loaded onto an agarose gel. The correct bands were cut out and extracted. Plasmid fragments of pIG23_03 and pIG23_04 were first assembled to form the sensor backbone with Gibson Assembly and AQUA cloning, according to the protocols. A transformation was done and 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, 50 mg/mL spectinomycin ) and sent for sequencing for correct insertion and no mutation.
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Then with the sensor backbone as a template the PCR was performed to add the correct overhangs to insert the riboswitch between lacI promoter and <i>lacI</i>. A Dpn1 digest was done at 37°C for an hour, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted.  
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The PCR for the riboswitch was also performed, subsequently, the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted.
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Riboswitch and sensor backbone were assembled via Gibson Assembly and AQUA cloning according to the protocols. A transformation was done and 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, 50 mg/mL spectinomycin ) and sent for sequencing for correct insertion and no mutation.
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Latest revision as of 00:59, 12 October 2023


piG_K12BSb (LacIprom_riboK12_T7RBS_lacI_trcProm_sfGFP)


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 215
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

If we had placed the sfGFP/marker protein directly after the riboswitch, its negative regulation in the presence of AdoCbl would have resulted in a decrease in fluorescence. In this BioBrick the riboswitch is placed in front of a repressor gene (lacI) which inturn suppresses sfGFP expression. If the riboswitch is triggered by binding of AdoCbl, the repressor expression is stopped leading to a detectable fluorescent signal.


Cloning of piG_K12BSb

Plasmid piG_K12BSa (BBa_K4604026) functioned as a template for a PCR to insert a stronger RBS from the TetR promoter (modified T7 RBS) directly behind the riboswitch. Gibson primer were designed to have the RBS as their overhang/complementary region. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 72°C and an elongation time of 6 minutes. A Dpn1 digest was done at 37°C overnight, afterwards the DNA was loaded onto an agarose gel. The correct bands were cut out and extracted. Gibson Assembly and AQUA cloning was used according to the protocols to assemble the plasmid. A transformation was done. As the change in the plasmid is only a RBS with a length of merely 14 bp a colony PCR was not possible to perform as screening methode, because the difference in length wouldn't have been noticeable on the gel. Instead DNA of several colonies was isolated from overnight cultures (5mL LB-medium, 50 mg/mL spectinomycin) and sent for sequencing to check for correct insertion and no mutation.