Team:SJTU-BioX-Shanghai/Results/Test/Overall

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Now it is the very time to test the whole system!! :)
Now it is the very time to test the whole system!! :)
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<br><br><br>
=Overall System=
=Overall System=
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----
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<br>
=='''Blue System'''==
=='''Blue System'''==
<html><img src="/wiki/images/c/cf/Blue_light_principle_sjtu.gif" width="600"/></html>
<html><img src="/wiki/images/c/cf/Blue_light_principle_sjtu.gif" width="600"/></html>
<br>
<br>
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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 mRFP under control of blue light sensor.
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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.
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After careful discussion, we decide to take up a <font size=4.5>'''three-step'''</font> manner:
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==Plasmid mRFP test==
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'''Light Gradient'''
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<br>
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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).
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=='''First Test'''==
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'''Ensure Similar OD'''
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'''GOAL''': To '''discover a coarse range of adjustable region'''.
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<br>
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<p>
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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..
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We set up a relatively '''sparse gradient''' of electric intensities on the User Interface. And corresponding blue lights would be produced into culture tubes, inside which there are 5mL culture of E. coli cells bearing above mentioned plasmids.
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</p>
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<p>
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We check the result at regular intervals.
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</p>
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<p>
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After a series of careful experiments and data collections, we successfully confirmed that:
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* when electrical intensities are '''under 40''', these electrical intensities are in a '''nice positive relationship''' with the mRFP amount.
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* And when electric intensities '''exceeds 50''', the system becomes saturated.
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</p>
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<p>
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As soon as we have acquired this suitable range of UI inputs, we immediately continue to our Second Test, in which we would like to further verify this range, deriving a detailed relation curve in this region.
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</p>
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=='''Second Test'''==
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'''Measure mRFP Expression'''
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'''GOAL''': To '''further verify this adjustable range, deriving a more accurate relation curve in this region'''.
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According to results from our first test, we set up a finer gradients:
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<br>
<br>
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from '''0 to 35''', with an '''interval of 5'''.
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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.
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[[File:SJTU13mRFP.png]]
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'''Figure 2. Relationship between mRFP expression and light intensity.'''<br> (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.<br> (b). A photo of the experiment result in Figure 2a. The red color gradient of mRFP is observable even by naked eye.
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'''Properties for Precise Regulation'''
<br>
<br>
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All other conditions are the same with the First Test.
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'''·''' 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.  
<br>
<br>
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We expect a good positive correlation between the amount of mRFP, the output, and the our UI inputs.
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'''·''' 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.  
<br>
<br>
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But surprisingly, the result is '''a little bit different from our prediction''':
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'''·''' 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.
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[[File:blueblueblue111.jpg|1200px]]
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==Endogenous fadD Test==
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'''Genome-residing Gene?'''
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<br>
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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.
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<br>
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Unlike plasmid genes, genome-residing genes are generally single-copied, thus may behave differently under regulation.
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[[File:Second test of overall sjtu.png|1200px]]
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'''Redirect Regulation'''
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<br>
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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.
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The correlation between the output is '''not always positive in this range''':
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'''Ensure Similar OD'''
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* The correlation is indeed positive between 15 and 35. And to our delight, the relationship is approximately '''LINEAR'''.
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<br>
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* But the correlation is, '''on the contrary, negative''' under 15.
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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.
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Together with the case study above, we have set up strict BLANK controls: these control strains do not contain gRNAs. We paralleled the control group with the case group.
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'''Assay fadD mRNA Amount with RT-PCR'''
<br>
<br>
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We proved that, mRFP amount in the control group would not vary significantly with the increase of light intensity (that is, with the increase of our UI input), which is a strong evidence that those relationships we observe in case groups is due to the correct function of our overall system, not just a effect of varied lights.
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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.
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[[File:qwqwqwqw.jpg|1200px]]
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[[File:SJTU13fadD.png]]
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[[File:Second control group of overall sjtu.png|1200px]]
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'''Figure 3. Relationship between fadD transcription and light intensity.'''
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<br>
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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.
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Such linear relationship demonstrates a bright future of the application of our system, but this range is a little bit narrow. Holding the wish of widening the linear relation range, we test for the third time. Also it will be more persuasive to say that our system is reliable due to all the repeat tests give the right results.
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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:
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<br>
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=='''Third Test'''==
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'''·''' The regulation range is wide;
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<br>
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'''·''' The increase is steady (R square=0.924);
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<br>
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'''·''' The system performance is relatively robust.
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Based on the previous tests and results we change the testing region to 15 to 45, and we hope to get the linear relationship. Data collecting work shows a good result again.
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==Conclusion of Blue System Evaluation==
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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.
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[[File:third group of overall sjtu.png|1200px]]
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Up to now we have proved that the overall system can function well and we have already found out the range in which there will be a linear relationship between input parameters and the targeted gene amount, i.e. between the light intensities and mRFP amount. Based on our versatile and easy-to-use system, further test related to new application on genomic level about metabolic regulation is going to be performed.
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<br><br><br>
=='''Red System'''==
=='''Red System'''==
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To verify the validity of our red system, we use luciferase as a reporter gene. 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.
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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.
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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.
[[File:Red luciferase result.png|1000px]]
[[File:Red luciferase result.png|1000px]]
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<br><br><br>
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<html>
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<h1 style="color:grey;">References</h1>
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<p style="color:grey;">
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<br>
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OHLENDORF, R., VIDAVSKI, R. R., ELDAR, A., MOFFAT, K. & M GLICH, A. 2012. From dusk till dawn: One-plasmid systems for light-regulated gene expression. Journal of Molecular Biology, 416, 534-542.
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</p></html>

Latest revision as of 01:38, 17 January 2014


Verified Components


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

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.

SJTU13mRFP.png

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.

Properties 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.

SJTU13fadD.png

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 (R square=0.924);
· The system performance is relatively robust.

Conclusion of Blue System Evaluation

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.




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.

Red luciferase result.png


References


OHLENDORF, R., VIDAVSKI, R. R., ELDAR, A., MOFFAT, K. & M GLICH, A. 2012. From dusk till dawn: One-plasmid systems for light-regulated gene expression. Journal of Molecular Biology, 416, 534-542.