Difference between revisions of "Team:Tec-Monterrey/Improve"
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For the correct mechanism of E-coding in sensing contaminants of the environment and integrating an analog message in the bacteria genome, previous parts found in the iGEM registry were used and optimized. This includes the SCRIBE system, previously characterized by iGEM Team UIUC 2015 (BBa_K1681001) based on the work of Lu and Farzadfard and the Pyear-GFP composite which express fluorescence in presence of nitrate ions (BBa_K381001). | For the correct mechanism of E-coding in sensing contaminants of the environment and integrating an analog message in the bacteria genome, previous parts found in the iGEM registry were used and optimized. This includes the SCRIBE system, previously characterized by iGEM Team UIUC 2015 (BBa_K1681001) based on the work of Lu and Farzadfard and the Pyear-GFP composite which express fluorescence in presence of nitrate ions (BBa_K381001). | ||
<div class="body-subtitle">SCRIBE(BsaI): BBa_K1681001 </div> | <div class="body-subtitle">SCRIBE(BsaI): BBa_K1681001 </div> | ||
| − | This part holds a single SCRIBE cassette induced by a lac operon. The cassette holds an editable retron sequence,the expression of a reverse transcriptase and a beta recombinase for DNA recombination into the bacteria's genomic DNA. iGEM Team UIUC did not include results regarding its specific parts, however, they did obtain successful recombination of a target DNA into the genome with a variant of this part with the same principle. From their work, as well as that from Farzadfard, a different design for this construct is proposed, where is it | + | This part holds a single SCRIBE cassette induced by a lac operon. The cassette holds an editable retron sequence,the expression of a reverse transcriptase and a beta recombinase for DNA recombination into the bacteria's genomic DNA. iGEM Team UIUC did not include results regarding its specific parts, however, they did obtain successful recombination of a target DNA into the genome with a variant of this part with the same principle. From their work, as well as that from Farzadfard, a different design for this construct is proposed, where is it adapted for the implementation of Cas1 and Cas2 proteins, instead of a recombinase, for the integration of target DNA to the bacteria's genome. |
<div class="body-subtitle">Pyear-GFP composite:BBa_K381001</div> | <div class="body-subtitle">Pyear-GFP composite:BBa_K381001</div> | ||
This part holds a cassette for the expression of GFP in the presence of nitrates. The original composite part consists of a promoter (Pyear) silenced by a protein NsrR, which is natively produced by Escherechia coli and a cassette for expression of a GFP. NsrR protein is inhibited by nitrate ions, so that in its presence the cassette expresses the GFP and fluorescence is observed. The problem from this construct is that under basal conditions without the presence of nitrate ions, there is leaking activity in the promotor which produces GFP. To solve this problem, a new cassette for the expression of NsrR was made to overexpress this inhibition factor and reduce the leaking activity in the promotor region. | This part holds a cassette for the expression of GFP in the presence of nitrates. The original composite part consists of a promoter (Pyear) silenced by a protein NsrR, which is natively produced by Escherechia coli and a cassette for expression of a GFP. NsrR protein is inhibited by nitrate ions, so that in its presence the cassette expresses the GFP and fluorescence is observed. The problem from this construct is that under basal conditions without the presence of nitrate ions, there is leaking activity in the promotor which produces GFP. To solve this problem, a new cassette for the expression of NsrR was made to overexpress this inhibition factor and reduce the leaking activity in the promotor region. | ||
Revision as of 21:10, 17 October 2018
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Previous Parts
For the correct mechanism of E-coding in sensing contaminants of the environment and integrating an analog message in the bacteria genome, previous parts found in the iGEM registry were used and optimized. This includes the SCRIBE system, previously characterized by iGEM Team UIUC 2015 (BBa_K1681001) based on the work of Lu and Farzadfard and the Pyear-GFP composite which express fluorescence in presence of nitrate ions (BBa_K381001).
SCRIBE(BsaI): BBa_K1681001
This part holds a single SCRIBE cassette induced by a lac operon. The cassette holds an editable retron sequence,the expression of a reverse transcriptase and a beta recombinase for DNA recombination into the bacteria's genomic DNA. iGEM Team UIUC did not include results regarding its specific parts, however, they did obtain successful recombination of a target DNA into the genome with a variant of this part with the same principle. From their work, as well as that from Farzadfard, a different design for this construct is proposed, where is it adapted for the implementation of Cas1 and Cas2 proteins, instead of a recombinase, for the integration of target DNA to the bacteria's genome.
Pyear-GFP composite:BBa_K381001
This part holds a cassette for the expression of GFP in the presence of nitrates. The original composite part consists of a promoter (Pyear) silenced by a protein NsrR, which is natively produced by Escherechia coli and a cassette for expression of a GFP. NsrR protein is inhibited by nitrate ions, so that in its presence the cassette expresses the GFP and fluorescence is observed. The problem from this construct is that under basal conditions without the presence of nitrate ions, there is leaking activity in the promotor which produces GFP. To solve this problem, a new cassette for the expression of NsrR was made to overexpress this inhibition factor and reduce the leaking activity in the promotor region.
Improvement Parts
In the integration of a DNA spacer into bacteria's CRISPR loci by means of Cas1-Cas2 integrase activity, the target DNA sequence to be deliberately inserted must be produced. This sequence is generated following the same basis as the SCRIBE system: a reverse transcriptase that retrotranscribes a retron sequence. Improved Part BBa_K2761010 contains two cassettes responsible for the production of this DNA message.
Inducible message generator (RT/msr-msd)
To adapt this DNA sequence generation for the integration with Cas1-Cas2 proteins, the target DNA in the msd region of the retron must first contain a PAM sequence, recognizable by the Cas proteins. Along with the PAM sequence, the rest of the msd must be a specific sequence to be associated with the specific inductor that triggers the system.Additionally, the reverse transcriptase is set to be expressed in a separate cassette as the retron, both under a same promoter, so as to avoid false positives resulting from leaking.
Characterization
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