Difference between revisions of "Part:BBa K2996011"
 
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<partinfo>BBa_K2996011 short</partinfo>
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<partinfo>BBa_K2996007 short</partinfo>
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This composite part is the core of our pRead plasmid in information storage device, where input signal is recorded as a permanent spacer.  
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<br>Integration cassette in pRead consists of a leader and two repeats interspaced by a spacer. We name it RSRL array, whicch is fused downstream to the complete coding sequence of an out-of-frame EGFP gene (eGFP +1).
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<br>Upon addition of inducer, spacer adaptation by Cas1/2 will result in an addition of 61 base pairs into the RSRL array. This moves the stop codon out of frame and EGFP into the ORF. Expression of in-frame EGFP is further induced by IPTG under tac promoter, so that recorded information can be read whenever desired through addition of IPTG.
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<center>{{#tag:html|<img style="max-width: 60%" src="https://static.igem.org/mediawiki/parts/3/34/T--SJTU-BioX-Shanghai--wet_lab-before.png" alt="" />}}</center>
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<center><b>Figure 1.</b> <i>Schematic representation of pRead before induction.</i> </center>
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<center>{{#tag:html|<img style="max-width: 60%" src="https://2019.igem.org/wiki/images/a/ad/T--SJTU-BioX-Shanghai--wet_lab-after.png" alt="" />}}</center>
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<center><b>Figure 2.</b> <i> Schematic representation of pRead after induction</i> </center>
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===Protocol===
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<b>1.Induction</b>
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<br>1.Inoculate 1% overnight bacterial culture in Erlenmeyer flask.
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<br>2.Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3).
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<br>3.Add IPTG to final concentration of 100 ng/μL.
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<br>4.Incubate overnight at 20℃.
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<b>2. Fabrication of alginate hybrid hydrogel</b>
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<br>Material: 5mg/mL sodium alginate, 50mg/mL tetracycline, 0.1M CaCI2, 100mg/mL PDB dissolved in DMSO, 1M IPTG.
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<br>Protocol:
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<br>1.Centrifuge the bacteria culture in its early exponential stage at 4000rpm for 3min.
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<br>2.Discard supernatant and resuspend it with 5mg/mL alginate to reach OD 0.2.
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<br>3.Seed 100μL alginate mixed with bacteria into 96 well plate.
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<br>4.Add 40μL crosslink reagent 0.025M CaCl2 or 1.25mg/mL PDB to each well.
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<br>5.Add 10μL tetracycline of 1mg/mL to each well.
  
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The ribosome-binding site and the translation initiation codon of this reading device are indicated. Translation of transcripts generated from the tac promoter will stop at the leader, for those two stop codons.
 
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The wrong reading frame and existing stop codon together will insure no expression of the florescent protein in the original situation.
 
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<li style="display: inline-block;"> [[File:T--SJTU-BioX-Shanghai--wet_lab-before.png|thumb|none|500px|'''Schematic representation of pRead before induction.''']]
 
  
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===Experiments and Results===
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<b> 1.System test </b>
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<br>As has been demonstrated before, our initial design incorporates a PAM-protospacer, Cas1-Cas2 complex protein and a modified CRISPR array. For starters, the input/ output mechanism of co-transformants containing plasmid pTrig BBa_K2996012, pRec BBa_K2996007 and pRead BBa_K2996011.
 +
Incubated culture was induced and trisected for different induction patterns. Positive samples were induced with both tetracycline and IPTG. Negative samples were induced with tetracycline only. Control samples were induced with IPTG.
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<center>{{#tag:html|<img style="max-width: 65%" src="https://static.igem.org/mediawiki/parts/4/46/T--SJTU-BioX-Shanghai--WYQ3.png" alt="" />}}</center>
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<center><b>Figure 1.</b> <i>Fluorescence of cells harboring pTrig, pRec and pRead</i> </center>
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Cells induced with IPTG and tetracycline showed significant increase in fluorescence value, indicating successful signal input.
  
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<div><ul> <li style="display: inline-block;"> [[File:T--SJTU-BioX-Shanghai--wet_lab-after.png|thumb|none|600px|'''Schematic representation of pRead after induction.''']]
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<b> 2.Test on alginate-PDB hybrid gel</b>  
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<br>We prepared a large amount of hybrid-hydrogel (48 units for each group) and conducted experiments through 50 hours.Cells induced with tetracycline showed significant increase in fluorescence value, indicating successful signal input. Around 40 hours post induction, fluorescence of uninduced samples decreased by two fold due to quenching.
 +
For pre-stored information to be extracted at any time, we added IPTG for the second round of induction, where the expression of EGFP can be promoted.  
  
 +
<center>{{#tag:html|<img style="max-width: 70%" src="https://static.igem.org/mediawiki/parts/b/b1/T--SJTU-BioX-Shanghai--WYQ2.png" alt="" />}}</center>
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<center><b>Figure 2.</b> <i>Fluorescence intensity change in a time course with E. coli grown in alginate-PDB.</i> </center>
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The significant increase in fluorescence intensity confirmed spacer adaption and our design as a re-readable biostorage device.
  
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<b> 3.QR code imitation</b>
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<br>Our engineered bacteria were encapsulated in alginate-PDB hydrogel and seed on each well. According to the designed pattern (for detailed informantion, see our model), we added tetracycline to black dots, as signal input and detected its florescence intensity after overnight culture to read the stored information. 
  
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<center>{{#tag:html|<img style="max-width: 95%" src="https://static.igem.org/mediawiki/parts/e/e5/T--SJTU-BioX-Shanghai--result_2.6.png" alt="" />}}</center>
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<center><b>Figure 3.</b> <i> Original signal input pattern </i> </center>
  
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Upon information input, that is addition of corresponding inducer, spacer adaptation from cas1/2 will result in an addition of 61 base pairs, 33bp new spacer and 28bp replicated repeat, into the integration cassette. Stop codon will be moved out of frame, while EGFP in the open reading frame.  
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<center>{{#tag:html|<img style="max-width: 80%" src="https://static.igem.org/mediawiki/parts/1/16/T--SJTU-BioX-Shanghai--result_2.7.png" alt="" />}}</center>
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Thus, successful translation of EGFP is our desired information output signal, observed by eyes and microplate reader.
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<center><b>Figure 4.</b> <i> Florescence intensity after overnight induction </i> </center>
  
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<center>{{#tag:html|<img style="max-width: 80%" src="https://static.igem.org/mediawiki/parts/d/d6/T--SJTU-BioX-Shanghai--result_2.8.png" alt="" />}}</center>
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<center><b>Figure 5.</b> <i>Signal output </i> </center>
  
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===Experiment and Result===
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All the results indicated the ‘iGEM-SJTU-BioX-Mulan’ information has been successfully encoded in two 96-well plate.  
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1.Overnight cultures of co-transformants were transferred and incubated at 37℃, 200rpm until cells grew into exponential stage.
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2.Samples were then trisected for different induction patterns.
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3.Positive samples were induced with both tetracycline and IPTG. Negative samples were induced with tetracycline only. Blank samples were not induced as control group. Samples were then incubated overnight at 20℃ for 16h.
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4.OD600 and fluorescence data were acquired from microplate reader, with excitation wavelength at 485nm and emission wavelength at 528nm. OD600 of all samples were adjusted to 0.5 before fluorescence measurement.
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Cells induced with IPTG and tetracycline showed significant increase in fluorescence value, indicating successful signal input. However, induction of sole tetracycline displayed a similar effect, indicating ineffectiveness of IPTG induction. This is due to high base expression of tac promoter, confirmed by our further experiments.
 
  
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Microscope
 
  
  

Latest revision as of 16:03, 26 November 2019


pTet upstream of cas1/2
Integration cassette in pRead consists of a leader and two repeats interspaced by a spacer. We name it RSRL array, whicch is fused downstream to the complete coding sequence of an out-of-frame EGFP gene (eGFP +1).
Upon addition of inducer, spacer adaptation by Cas1/2 will result in an addition of 61 base pairs into the RSRL array. This moves the stop codon out of frame and EGFP into the ORF. Expression of in-frame EGFP is further induced by IPTG under tac promoter, so that recorded information can be read whenever desired through addition of IPTG.


Figure 1. Schematic representation of pRead before induction.


Figure 2. Schematic representation of pRead after induction


Protocol

1.Induction
1.Inoculate 1% overnight bacterial culture in Erlenmeyer flask.
2.Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3).
3.Add IPTG to final concentration of 100 ng/μL.
4.Incubate overnight at 20℃.

2. Fabrication of alginate hybrid hydrogel
Material: 5mg/mL sodium alginate, 50mg/mL tetracycline, 0.1M CaCI2, 100mg/mL PDB dissolved in DMSO, 1M IPTG.
Protocol:
1.Centrifuge the bacteria culture in its early exponential stage at 4000rpm for 3min.
2.Discard supernatant and resuspend it with 5mg/mL alginate to reach OD 0.2.
3.Seed 100μL alginate mixed with bacteria into 96 well plate.
4.Add 40μL crosslink reagent 0.025M CaCl2 or 1.25mg/mL PDB to each well.
5.Add 10μL tetracycline of 1mg/mL to each well.


Experiments and Results

1.System test
As has been demonstrated before, our initial design incorporates a PAM-protospacer, Cas1-Cas2 complex protein and a modified CRISPR array. For starters, the input/ output mechanism of co-transformants containing plasmid pTrig BBa_K2996012, pRec BBa_K2996007 and pRead BBa_K2996011. Incubated culture was induced and trisected for different induction patterns. Positive samples were induced with both tetracycline and IPTG. Negative samples were induced with tetracycline only. Control samples were induced with IPTG.

Figure 1. Fluorescence of cells harboring pTrig, pRec and pRead

Cells induced with IPTG and tetracycline showed significant increase in fluorescence value, indicating successful signal input.

2.Test on alginate-PDB hybrid gel
We prepared a large amount of hybrid-hydrogel (48 units for each group) and conducted experiments through 50 hours.Cells induced with tetracycline showed significant increase in fluorescence value, indicating successful signal input. Around 40 hours post induction, fluorescence of uninduced samples decreased by two fold due to quenching. For pre-stored information to be extracted at any time, we added IPTG for the second round of induction, where the expression of EGFP can be promoted.

Figure 2. Fluorescence intensity change in a time course with E. coli grown in alginate-PDB.

The significant increase in fluorescence intensity confirmed spacer adaption and our design as a re-readable biostorage device.

3.QR code imitation
Our engineered bacteria were encapsulated in alginate-PDB hydrogel and seed on each well. According to the designed pattern (for detailed informantion, see our model), we added tetracycline to black dots, as signal input and detected its florescence intensity after overnight culture to read the stored information.

Figure 3. Original signal input pattern
Figure 4. Florescence intensity after overnight induction
Figure 5. Signal output

All the results indicated the ‘iGEM-SJTU-BioX-Mulan’ information has been successfully encoded in two 96-well plate.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 252
    Illegal BamHI site found at 978
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]