Part:BBa_K2558206

CRISPRi device with Ptac expressed gRNA and Anderson strong (J23100)- expressed lacI

This construct is the CRISPRi device, which is composed of dCas9, Ptac controlled gRNA targeting PHIF repressible promotor (BBa_K1725000) and constitutively (BBa_J23100, Anderson strong promotor) expressed lacI protein. We designed this device along with K2558207 and K2558208, which are the same except for the strength of the constitutive lacI expression. We aim to test the repression efficiency of our CRISPRi system with the help of PHIF repressible promotor.

Usage and Biology

After we clarified the correlation between lacI dosage and its induction efficiency, we furthered the experiments by adding CRISPRi to the system. Instead of sfGFP, the IPTG induction devices with different levels of lacI generator is now going to produce a gRNA that targets the promotor of a constitutive sfGFP generator (BBa_K2558206, BBa_K2558207, BBa_K2558208). By adding IPTG, we can induce the transcription of gRNA. Binding with constantly expressed dCas9, gRNA is going to inhibit the expression of sfGFP. We will be able to observe how this process can operate under different levels of lacI expression.

Results

In this experiment, the expression of sfGFP was inhibited by IPTG induced CRISPRi system. (Figure.1) Without CRISPRi the Fluorescence/OD600 increased steadily. With medium lacI expression, the addition of IPTG caused the fluorescence intensity to decrease at three hours after addition. With different IPTG concentration the final sfGFP fluorescence intensity remained approximately the same, which is consistent with previous IPTG induction results. However, with weak lacI expression, sfGFP expression was inhibited from the beginning. With the direct IPTG induction experiment we performed before, we hypothesize that with the weak Anderson promotor, basal gRNA expression will be above the threshold that is required for suppressing the sfGFP expression.[1]

Figure.1. The effect of varied lacI-LVA (BBa_C0011) promotor strength on IPTG induction of CRISPRi. The Y axis values are fluorescence per OD600 standardized with fluorescence per OD600 value of each test group at Time=0, IPTG=0. The promotors we used are from the Anderson collection: BBa_J23110 for medium expression (orange), BBa_J23114 for weak expression (blue).

Protocol

The protocol is as follows:

1. Transform the plasmids into E. coli DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml M9 medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6。
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 3, 5, 10 and 20 mM. Fresh M9 medium serves as blank control. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 of each well every 30 minutes.
5. Each group should be repeated for at least 3 times.

Reference

[1] Mückl A, Schwarz-Schilling M, Fischer K, Simmel FC. “Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach.” PLoS One. 2018 Sep 11;13(9):e0198058. doi: 10.1371/journal.pone.0198058. eCollection 2018.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 310
  Illegal NheI site found at 333
  Illegal NheI site found at 1466
  Illegal NheI site found at 4626
  Illegal NheI site found at 4649
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5830
  Illegal BamHI site found at 3745
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]