Part:BBa_K2996011

RSRL-eGFP downstream of pTac

This composite part is the core of our pRead plasmid in information storage device, where input signal is recorded as a permanent spacer. An original pRead contains a CRISPR array (We name it RSRL), which consists of 69 bp leader and one spacer flanked by two repeats. Integration cassette is put upstream of the complete coding sequence of an out-of-frame EGFP gene (EGFP +1). Translation of transcripts generated from the tac promoter will stop at the leader, for those two stop codons. The wrong reading frame and existing stop codon together will prevent expression of the EGFP in the original situation.

Figure 1. Schematic representation of pRead before induction.

Upon information input, that is addition of corresponding inducer, spacer adaptation from cas1-cas2 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. Thus, successful translation of EGFP is our desired information output signal, observed by eyes and microplate reader.

Figure 1. Schematic representation of pRead after induction.

Protocol

1.Induction
1.Overnight cultures of co-transformants were transferred and incubated at 37℃, 200rpm until cells grew into exponential stage.
2.Samples were then trisected for different induction patterns.</br>
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.
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.

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 < a href="https://parts.igem.org/Part: BBa_K2996011"> BBa_K2996011</ a>. 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, click链接到model他们那个界面), 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]