Team:SJTU-BioX-Shanghai/Measurement
Measurement
Fluorescence & results
CRISPRa
In our constructed CRISPR-activation system, we use the weak promoter J23117 to induce the expression of fluorescent proteins. The target was set at the upstream of the RFP reporter’s promoter and the lure was planted in front of the eGFP reporter’s promoter.
In the absence of dCas9-ω, J23117 hardly expresses the RFP/eGFP reporter gene due to its weak initiation capability. When dCas9-ω is present, if sgRNA guides dCas9-ω to combine with the target region, the RFP reporter gene expression will be activated, thus showing red fluorescence. When the lure area is combined, the eGFP reporter is activated to show green fluorescence.
Therefore, the signal of red fluorescence corresponds to the on-target result of dCas9, while the signal of green fluorescence corresponds to the off-target result. In the process of flow cytometry sorting, dCas9 with strong red signal and weak green signal is expected.
CRISPRi
In our constructed CRISPRi (CRISPR-Inhibition) system, we use arabino-induced promoter to induce downstream reporter genes (fluorescent proteins).In this system, we choose to insert target between the promoter of the upstream target reporter gene (RFP) and the RBS sequence, and lure between the target reporter gene (eGFP) and the RBS sequence. We use arabinose to induce the expression of RFP and eGFP reporter genes.
The expression of RFP or eGFP would be interfered if dCas9 is combined with target or lure sequence. Therefore, the strong red/green fluorescence signal indicates that dCas9 does not combine target/lure sequence, while the weak fluorescence signal indicates that dCas9 combined target/lure sequence and played the inhibitory role of dCas9. dCas9 with weak read and strong green fluorescence signal will be selected by flow cytometry to obtain the desired dCas9.
Microplate reader
In order to quantify the fluorescence signal, we use microplate reader to measure the fluorescence intensity of RFP and eGFP. The absorption wavelength and emission wavelength of RFP and eGFP are 555/583nm and 483/507nm respectively. The fluorescence characterization of RFP and eGFP was detected by a multifunctional microplate reader, and the fluorescence intensity can read in specified values. The stronger the fluorescence intensity, the greater the measured value.
In the CRISPRa system, what we ultimately need is dCas9, which can produce a strong red fluorescence and weak green fluorescence effect in the gene circuit. In the microplate reader, it’s that value of red fluorescence is large and that of green fluorescence is small.
In the CRISPRi system, what we ultimately need is dCas9, which can generate strong green fluorescence and weak red fluorescence in the loop. The numerical expression measured by the microplate analyzer is that the green fluorescence value is large and the red fluorescence value is small.
Flow cytometry
In our screening process, after transforming mutation plasmid library into the screening of strain, each individual bacteria may contain different the plasmid type, have different dCas9 expression, and have different targets. The screening on individual won’t be very efficient if fluorescence detection is only carried out on the strain level, so we decided to use flow cytometry instrument as a better screening instrument.
Flow cytometry can detect the fluorescence expression level of individual bacteria. We used APC channel to detect the intensity of red fluorescence and FITC channel to detect the intensity of green fluorescence. We can quantify the fluorescence protein characterization level of a single bacteria according to the detection results, so as to obtain the distribution of a whole batch of bacteria according to the fluorescence characterization level.
We used non-mutated dCas9 as a control, with its fluorescence level represented in the CRISPRa/CRISPRi system strains as the screening criteria. At the same time, we also used the blank MG1655 strain without plasmid as the blank control, so as to distinguish the standard lines with or without fluorescence characterization.
In the CRISPRa system, what we need is the dCas9 that can increase the level of red fluorescence represented by RFP and decrease the level of green fluorescence represented by eGFP. With this screening standard, we can select the bacteria with both RFP representing red fluorescence level above the screening standard and eGFP representing green fluorescence level below the screening standard, and we can see the proportion of these bacteria in the total number of bacteria.
In the CRISPRi system, what we need is dCas9 that can increase the eGFP represented green fluorescence level and decrease the RFP represented green fluorescence level. With the screening standard, we can select the bacteria with both eGFP representing green fluorescence level above the screening standard and RFP representing red fluorescence level below the screening standard, and we can see the proportion of these bacteria in the total number of bacteria.
By analyzing the fluorescence characterization level of cells in the flow cytometer, we can clearly see that compared with the unmutated dCas9, the on-target and off-target effects of the mutated dCas9 can be obtained, and then the bacteria can be sorted according to our screening criteria, so as to obtain the mutant dCas9, which meets our the requirements.
SJTU-BioX-Shanghai
contact us
Contact us: sjtuigem@gmail.com or sjtuigem@gmail.com
Bio-X Institute, Shanghai Jiao Tong University, Dongchuan Rd. 800
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