Part:BBa_K2996011

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 <a href="https://parts.igem.org/Part: BBa_K2996012">BBa_K2996012</a>, pRec<a href="https://parts.igem.org/Part: BBa_K2996007">BBa_K2996007</a> and pRead (BBa_K2996011 :https://parts.igem.org/Part: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]