Verified Components

So far we have evaluated the function of all three components in our Box, with all of them exhibiting favaroble performances:

• Verification of Luminous System
• Empirical Support of Light Sensors
• Verification of CRISPRi

Now it is the very time to test the whole system!! :)

Overall System

Blue System

Figure 1. As described in our [http://igem.bio-x.cn/Team:SJTU-BioX-Shanghai/Project/Light_sensor/Blue Project page], we have placed a gRNA that targets either mRFP or fadD under control of blue light sensor.

Plasmid mRFP test

Light Gradient
A gradient of ten different light intensities was established within the sensing range of YF1-FixJ-PFixK2, from zero to saturation (Ohlendorf et al., 2012).

Ensure Similar OD
But to avoid the discrepancy of growing phase among different experiment groups, bacteria are cultured in darkness to stationary phase (OD600 ≈ 2.2) before they are divided. This procedure takes about 24 hours. And the fluorescence intensity reaches about 28.245 a.u..

Measure mRFP Expression
Then after another 15 hours’ culture under different blue light intensities, fluorescence intensities are measured again. The result is shown in Figure 2a. Bar height represents the increase of fluorescence intensity between two successive measurements, which corresponds the production rate of mRFP. With the increase of light intensity, we observed a gradual ascend in the expression strength.

Figure 2. Relationship between mRFP expression and light intensity.
(a). Quantitative measurement of mRFP produc-tion under different light intensities. Bar height represents mRFP production in 15 hours under different light intensi-ties. Error bars shows the standard error (s.e.) of parallel groups. mRFP production gradually increases about one-fold.
(b). A photo of the experiment result in Figure 2a. The red color gradient of mRFP is observable even by naked eye.

Three properties make our sensor-CRISPRi suitable for precise regulation:
· Under optical saturation, mRFP is produced about twice as fast as it is in the darkness, indicat-ing a relatively wide range for adjusting regulation effect. And the regulation range can be further enlarged by intro-ducing additional gRNAs for the same target.
· The squared Pearson coefficient (R2) of linear fit is calculated to be 0.901. So the expression is stably accelerated as we lift up light intensity (even if the relationship is not strictly linear), making it easier for researchers to locate an optimal regula-tion.
· The variance (standard errors are represented as error bars in Figure 2a) is relatively small. So our system is stable, and a pre-determined working curve can be referenced in later experiments.

Endogenous fadD Test

Genome-residing Gene?
We next examined how sensor-CRISPRi acts on fadD. Even though favorable results have already been acquired in mRFP tests, we still need to verify that our system also works for genome-residing genes.
Unlike plasmid genes, genome-residing genes are generally single-copied, thus may behave differently under regulation.

Redirect Regulation
We replaced the base-pairing region of gRNA by inverse PCR to redirect sensor-CRISPRi onto this fadD. From this redirection, a bonus of CRISPRi can be observed: in case that researchers change their targets, all they need is to substitute a 20-nt sequence.

Ensure Similar OD
Bacteria are cultured in darkness to stationary phase (OD600 ≈ 2.0) before they are divided into different experi-ment groups. After another 15 hours’ culture under different blue light intensities, cell bodies are collected for RNA ex-traction.

Assay fadD mRNA Amount with RT-PCR
Real-Time PCR (RT-PCR) is applied to assay the amount of fadD mRNA. gapA, the E. coli house-keeping gene for glyceraldehyde-3-phosphate dehydrogenase (GADPH, EC 1.2.1.12), served as the internal reference(8). And in relative quantitation (comparative threshold method), we took wild type E. coli strain BL21 (DE3) as the control. The result is presented in Figure 3.

Figure 3. Relationship between fadD transcription and light intensity. Bar height represents the relative amount of fadD mRNA. Error bars shows the standard error (s.e.) of parallel groups. Transcription level gradually increases about one-fold.

The eligibility of sensor-CRISPRi in precise regulation is confirmed on this genome-residing gene. mRNA amount of fadD increases continuously and steadily when blue light exposure is enlarged. All three properties revealed in plasmid mRFP test are repeated here:
· The regulation range is wide; · The increase is steady (R2=0.924); · The system performance is relatively robust.

In conclusion, by serially connecting blue light sensor (YF1-FixJ-PFixK2) and CRISPRi, the expression of target gene can be quantitatively related to light signals. Therefore, sen-sor-CRISPRi can be applied where it is necessary to precisely regulate endogenous genes, e.g. in medical therapies and in metabolic optimization. Prospectively, for multiple gene targets in a complex pathway, the system can be readily extended. We can incorporate additional sensors that respond to light of different wavelengths to control additional gRNAs for these new targets. Actually we have already incorporated a red light sensor and a green light sensor(9), and performance test is undergoing. Our ultimate goal is the accurate and systematic interrogation of cellular activities.

Red System

We also successfully constructed a red system, which can regulate the expression of genes with different strength of red light. To verify the validity of our red system, we use luciferase as a reporter gene. The protein luciferase can emit light with the existence of luciferin.

We observed obvious difference of the strength of fluorecence between the sample with and without the radiation of red light, which means that we can get larger amount of products under stronger red light. We has repeated it for 4 times, and the results are the same. The results are chiefly as follows.