Team:SJTU-BioX-Shanghai/Results
Real-time off-target detection
Detection system
The detection of off-target incidence is aimed to be efficient, effective, and real-time. We introduced lure sequences as potential off-target sites taken from previous published papers or predicted by our own software and a report system that incorporates two fusion proteins with a mechanism similar to split luciferase assay.
In our design, firefly luciferase is split into an amino-terminal fragment and a carboxyl-terminal fragment, fused respectively to dCas9 and zinc finger protein to form NLuc-dCas9 and CLuc-DBD. With zinc finger bound to a specific sequence adjacent to lure, when NLuc-dCas9 further binds to lure, physical proximity of the N- and C- terminal fragments allows the luciferase activity to reconstitute, producing luminescent signals.
Result
dCas9 with sgRNA acts as the key of off-target incidence. We got part BBa_K1689010 from team Peking.
The signal of off-target incidence is given by the combined protein of C-luciferase and N-luciferase. N-luciferase is already fused with dCas9 in BBa_K1689010. Then the problem lies in how to construct a steady module that can signal the off-target incidence once the N-luciferase-dCas9 fusion protein binds to the “lure” sequence. We searched in the part list for a DNA binding protein, and finally we got part BBa_K2996503 from team Fudan, and we call it DBD.
To make the DNA for the fusion protein of C-luciferase-DBD, our strategy was to put the DNA of C-luciferase and the DBD together by overlap PCR.
In the first part of the task, we used primers that contains sequence for promoter J23119(forward primer) and GGGGS protein linker (reverse primer) to copy the sequence of C-luciferase, the desired DNA fragment is 542bp.
Fig. 1 PCR product of C-luciferase with J23119 upstream.(Lane1:product of Cluc;Lane 2:2000bp ladder)
On the other hand, we used primers that contains sequence for GGGGS protein linker (forward primer) and rrnB T1 terminator (reverse primer) for PCR of DBD, the desired DNA fragment is 421 bp.
Fig. 2 PCR product of DBD with rrnB T1 terminator downstream.(Lane1:product of DBD;Lane 2:2000bp ladder)
After obtaining the PCR products of C-luciferase and DBD, we used overlap PCR to merge the two fragments.
Fig. 3 overlap PCR product of C-luciferase and DBD.(Lane1:overlap PCR product;Lane 2:2000bp ladder)
We first got the backbone of pCDFDuet-1 for Gibson assembly.
Fig. 4 PCR product of pCDFDuet-1 backbone for Gibson assembly.(Lane1-Lane5:PCR product;Lane 6:5000bp ladder)
Sequentially, we assembled pCDFDuet-1 backbone and the overlap PCR product with Gibson assembly. DH5 alpha was then transformed with assembled product. We used the DBD primers to check the colonies on the selection plate.
Fig. 5 Result of colony PCR with DBD primers.(Lane2-Lane9,Lane11-Lane18:colony PCR results;Lane 1,Lane 10,Lane 19:2000bp ladder)
Future anticipation
Due to the fact that lure sequence and sgRNA pair during application, we decided to put the sgRNA production module upstream of the lure and DBD’s binding site. Once the users want to detect off-target incidence, they can transfer the plasmid into bacteria with N-luciferase and C-luciferase. The plasmid was synthesized as part BBa_K2996507. Thus, we successfully constructed the plasmids of the detection parts.
Up till now,we have achieved the construction of all the plasmids we need in this system,which means that our system can probably work to detect off-target incidence. However, our work went on with the future verification experiments.
In the future, we will continue the work of cotransformation. After cotransformation of the three plasmids, we will change the lure sequences by point mutation, which are selected from science literature. The detection of the system with every mutated lure sequence will be recorded by the intensity of reaction of luciferase. This is aimed to support data for computer modeling, and to test whether the detection system correspond to the off-target probabilities in the literature.
Deferred off-target detection: Transcription activation
Testing the transcription activation degree of mRFP
We obtained three kinds of transformants, each harboring (a)pResponse-RFP, (b)pResponse-RFP plus pActivator without sgRNA, (c)pResponse-RFP plus pActivator with sgRNA. Several colonies were subjected to induction protocol.
Below is the data acquired from microplate reader, with excitation wavelength at 584nm and emission wavelength at 607nm. Florescence/OD600 indicates the intensity of mRFP.
Fig. 1 Fluorescence intensity of three kinds transformants before and after tetracycline induction.
We can see a 4-fold increase in florescence intensity after tetracycline induction, compared to itself and negative groups. So, we are confident that the constructed RpoA-dCas9 transcription activator has the desired biological function.
Analyse how pLuxR and its downstream genes respond to AHL induction.
As mentioned before, the recording of off-target incidence can be achieved through this transcription activation device, with LuxI gene is under the control of this promotor. The increased expression of LuxI enzyme will synthesis enough AHL molecules, which can penetrate to the bacteria capable of information storage and act on promotor luxR. BBa_K2558001.
Analysis of pLux-repL copy number
We tried different AHL concentration from 0 to 10-6 M and measured copy number in the time course. Both absolute copy number and relevant copy number amplification compared to non-induction were calculated.
The result confirmed the small leakage of luxpR-HS and it also showed obvious responding to AHL, with the maximum effect reached at a concentration of 10-7 M at 3.5h of induction.
Fig. 2 change of absolute copy number under different induction concentration
Fig. 3 change of relative copy number under different induction concentration
Fig. 4 change of absolute copy number under different induction concentration
Fig. 5 change of relative copy number under different induction concentration
Analysis of Cas1-Cas2 expression
Two successful transformants harbouring pLux-cas, pLux-trig and pRead plasmids, positive strain (successful transformant which exhibit EGFP after tet induction) and control (E. coli BL21) are grown overnight at 30℃ with 10-7M AHL or 100ug/mL tetracycline.
Below is the result from SDS-PAGE analysis. Since Cas1-Cas2 complex is around 44 kDa, we believe it was successfully expressed in our engineered bacteria.
Fig. 6 SDS-PAGE analysis of Cas1-Cas2 complex.
Summary
pActivator, RpoA-dCas9 BBa_K2996701, can activate corresponding promotor. Also, we have constructed pLux-repL and pLux-cas with practical function, realizing record of off-target incident through transcription activation system.
The concept of off-target lure is applied for CRISPRa to activate downstream genes and trigger other responses.
Using overlap PCR, RpoA is linked to dCas9 through a modified flexible Linker 4 (FL4), which has been proved to be effectively separate bifunction proteins.
Now we got the transcription activator partBBa_K2996701.
Future work:Triggered responses
To facilitate measurements, we are going to use a reporter plasmid, with the mRFP gene under the control of a constitutive promoter (BBa_J23117) that is preceded by a sequence rich in NGG PAM sequences on the NT strand. Thus, florescence intensity can indicate the level of transcription activation.
To test the activation ability of our design, we first plan to use gRNA that complements the NT strand from at least 20nt upstream from -55 point (With the transcription start denoted +1, RNAP complex covers from -55 to +20.)
When dCas9 binds to lure and recruits RNA polymerase, promotor J23101 will be activated, thus increasing the expression of mRFP florescence.
As mentioned before, the recording of off-target incidence can be achieved through this transcription activation device, with LuxI gene is under the control of this promotor. The increased expression of luxI enzyme will synthesis enough AHL molecules, which can penetrate to the bacteria capable of information storage and act on promotor luxR.
CRISPR biostorage of off-target signal
Signal input and output
Cloning and Transformation
As has been demonstrated above, our initial design incorporates a PAM-protospacer, Cas1/2 protein and a modified CRISPR array. A proper storage device contains plasmid pTrig, pRec and pRead.
In fluorescence microscope, the induced biostorage device displays strong fluorescence. Fluorescence in cells without pTrig appears dim and no fluorescence is displayed in uninduced device. This confirms our success in input and output, and also the necessity of every part involved.
Fig.1 Fluorescence microscope results
We then tested storage device with different induction patterns. Devices induced with both IPTG and tetracycline displayed an ideal level of fluorescence, compared to control group that were not induced.
Fig.2 Fluorescence of cells harboring pTrig, pRec and pRead
Test for system robustness
Due to high base expression of tac promoter, EGFP is inevitably expressed upon signal input without IPTG induction. However, we can still achieve deferred output since plasmid pRead containing in-frame EGPF is always present in induced cells and can still be induced by IPTG. Cell culture induced by tetracycline was preserved at 4℃ while monitoring fluorescent intensity each 2 days to observe fluorescence quenching. After 3 days, IPTG induction was conducted to measure fluorescence.
Fig.3 Restored Fluorescence of cells harboring pTrig, pRec and pRead
On day 3, fluorescence of uninduced samples decreased by 2 fold due to quenching. After IPTG induction, however, fluorescence was restored. This indicates successful protospacer insertion and information storage.
Extension: hydrogel QR code
The optimal culture environment: test on alginate-PDB hybrid gel
We aim to make this CRISPR cell storage device just like compact disc. To realize this, we fixed bacteria chassis in microwell arrays where each confined cell is a recording unit.
Due to the change of culture environment, we tested specially fabricated hydrogel to encapsulate engineered E. coli, so as to gain best function of this storage system.
After a series of trials (click for detailed data), we decided to use sodium alginate aligned with 1,4-bi(phenylalanine-diglycol)-benzene (PDB) as culture media.
To begin with, we tested 4 different types of culture media, including 5mg/mL alginate cross linked with CaCI2, 5mg/mL alginate cross linked PDB, 5mg/mL PDB and LB broth, along with 2 different induction methods.
Fig. 5 Fluorescence intensity change with alginate-PDB
Fig. 6 Fluorescence intensity change with alginate-PDB
Fig. 7 Fluorescence intensity change with pre-mixed alginate-PDB
The volume of tetracycline (10mg/mL) didn’t affect the results too much and alginate cross-linked by CaCI2 or PDB were practical media for bacteria culture.
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
We have prepared a large amount of hybrid-hydrogel (48 units for each group) and conducted a new round of 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.
Fig. 8 Fluorescence intensity change in a time course with E. coli grown in alginate-CaCl2.
Fig. 9 Fluorescence intensity change in a time course with E. coli grown in alginate-PDB.
QR code imitation
In a practical way, with panels seeded with our engineered bacteria, information transformed into QR-code can be recorded by adding inducer to dots that are black. Information can be easily extract by scanning the plate by a microplate reader.
Our engineered bacteria were encapsulated in alginate-PDB hydrogel and seed on each well. According to the designed pattern (for detailed informantion, click HERE), we added tetracycline to black dots, as signal input and detected its florescence intensity after overnight culture to read the stored information.
Fig. 10 Original signal input pattern.
Fig. 11 Florescence intensity after overnight induction.
Fig. 12 Signal output
Summary
To sum up, our experiment confirmed that the designed system functioned properly. Information was stored upon tetracycline addition and could be read afterwards by adding IPTG, given that cells are viable and active.
Nevertheless, experiments confirmed that tac promoter is not the choice for EGFP expression control. Instead, strict promoters are preferred for this task, for example arabinose promoters.
References
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SJTU-BioX-Shanghai
Contact us: sjtuigem@gmail.com
Bio-X Institute, Shanghai Jiao Tong University, Dongchuan Rd. 800