Difference between revisions of "Part:BBa K3086009"

 
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<partinfo>BBa_K3086009 parameters</partinfo>
 
<partinfo>BBa_K3086009 parameters</partinfo>
 
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<p>Our overall project can be broken down into three components. (1) Verifying the SCRIBE system (2) Compare the efficiency of the SCRIBE system against using Cas9 as a selection tool (3) Using the Van promoter to make the SCRIBE system more efficient.</p>
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<h2>Verifying the SCRIBE system</h2>
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<p>For the first part of our project, we are testing to see if Fahim Farzadfard and Timothy K. Lu’s paper, “Genomically encoded analog memory with precise in vivo DNA writing in living cell populations” rings true in regards to whether the SCRIBE system works or not. In order to check for the SCRIBE system, we incorporated a target sequence that gave rise to specific antibiotic resistance. rpoB gives rise to rifampicin resistance and rpsL gives rise to streptomycin resistance. If the SCRIBE system is working correctly, then the target sequence was successfully incorporated into the chromosomal DNA of the <i>E.coli</i> cell. Therefore the host cell will express the corresponding antibiotic resistance.  </p>
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<h1>Rifampicin serial dilution plates</h1>
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<h3>Experiments 1-4</h3>
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<center><img src="https://2019.igem.org/wiki/images/a/aa/T--Florida--rif_plate_101103.jpeg"
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<center><font size="4">Dilution factor: 10<sup>-1</sup> to 10<sup>-3</sup></font></center>
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<center><img src="https://2019.igem.org/wiki/images/8/8b/T--Florida--rif_plate_104106.jpeg"
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style="
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<center><font size="4">Dilution factor: 10<sup>-4</sup> to 10<sup>-6</font></center>
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<p>We checked to see if the rpoB target sequence was incorporated into the pFF745 plasmid and whether it gave the host cells rifampicin resistance. This was proved by the growth of colonies on the rifampicin plates. <p>
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<h2>Experiment 1</h2>
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<p>The first experiment with E.coli represents the negative control therefore proving that pFF745 does not normally express rifampicin resistance. </p>
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<h2>Experiments 2, 3, and 4</h2>
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<p>Experiments 2, 3 and 4 are all E. coli with the rpoB target sequence. Since they were able to grow on the rifampicin plate, we can conclude the SCRIBE system successfully worked. </p>
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<p>The sequencing data shows that the rpoB target was successfully put into the plasmid. The sequencing reaction is on the bottom and the designed plasmid sequence is on the top. The gray bars at the top show that the whole sequence matches. <p>
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<center><img src="https://2019.igem.org/wiki/images/a/aa/T--Florida--rpob_sequence.png"
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style="
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width:950px;
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Revision as of 00:16, 22 October 2019


SCRIBE (RpoB) System

This is our validated part. SCRIBE( Synthetic Cellular Recorders Integrating Biological Events) is a tool that utilizes modified retrons that have been transformed into bacterial cells to produce single-stranded DNA in response to a certain stimulus. The ssDNA is incorporated into the genome using the replication system of the bacteria. This technique mutates the bacteria to have rifampicin resistance.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1877
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1877
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1282
    Illegal XhoI site found at 516
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1877
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1877
  • 1000
    COMPATIBLE WITH RFC[1000]


Our overall project can be broken down into three components. (1) Verifying the SCRIBE system (2) Compare the efficiency of the SCRIBE system against using Cas9 as a selection tool (3) Using the Van promoter to make the SCRIBE system more efficient.

Verifying the SCRIBE system

For the first part of our project, we are testing to see if Fahim Farzadfard and Timothy K. Lu’s paper, “Genomically encoded analog memory with precise in vivo DNA writing in living cell populations” rings true in regards to whether the SCRIBE system works or not. In order to check for the SCRIBE system, we incorporated a target sequence that gave rise to specific antibiotic resistance. rpoB gives rise to rifampicin resistance and rpsL gives rise to streptomycin resistance. If the SCRIBE system is working correctly, then the target sequence was successfully incorporated into the chromosomal DNA of the E.coli cell. Therefore the host cell will express the corresponding antibiotic resistance.

Rifampicin serial dilution plates

Experiments 1-4

Dilution factor: 10-1 to 10-3

Dilution factor: 10-4 to 10-6

We checked to see if the rpoB target sequence was incorporated into the pFF745 plasmid and whether it gave the host cells rifampicin resistance. This was proved by the growth of colonies on the rifampicin plates.

Experiment 1

The first experiment with E.coli represents the negative control therefore proving that pFF745 does not normally express rifampicin resistance.

Experiments 2, 3, and 4

Experiments 2, 3 and 4 are all E. coli with the rpoB target sequence. Since they were able to grow on the rifampicin plate, we can conclude the SCRIBE system successfully worked.

The sequencing data shows that the rpoB target was successfully put into the plasmid. The sequencing reaction is on the bottom and the designed plasmid sequence is on the top. The gray bars at the top show that the whole sequence matches.