Team:CGU Taiwan/Results
Results
Yeast With IL-8 Receptor
Toehold Switch As RNA Sensor
Result toehold secondary structure prediction
RESULTS
The pictures are the secondary structure prediction of four toehold switches that we design, which we want to use to sense the four biomarkers we chose to detect in oral cancer’s patients’ saliva. We use the website RNA structure :
(http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html)
to predict their secondary structures. These structures must contain hairpin structure, which could have the switch on-off mechanism to sense the designed target RNA and trigger RNA we to test its efficiency, and further the sensitivity and specificity of this detection method. Also, the secondary structure can’t be annealed randomly, and the linker and trigger RNA can’t be complementary. These four secondary structures of the sequences are correspond with our expectation. Then we can further do the experiment of construction of toehold switches.
Methods
Toehold switch sequence design
Seven oral cancer biomarkers are found in a previous research [3], and four of them are chosen to be the target of our project, which are IL-8, IL-1β, dual specificity phosphatase 1(DUSP1), and spermine N1-acetyltransferase(SAT). We design our toeholds by getting our target sequences from the NCBI website, and then we define and design our target region into 30 base pairs. It is very important that it contains about equal amounts of ATCGs, and we need to make sure they don’t match with human genome; also, they cannot contain restriction enzyme sites in the sequences. When we complete the stem loop, we add luciferase, which is our reporter, at the end of the sequences. Finally, predict the structure with RNA Structure website (http://rna.urmc.rochester.edu/RNAstructureWeb) to confirm the secondary structure of our toehold switches.Biobricks assemble
To construct the sensor and test its quality, we built two plasmids which contain toehold switches and triggers respectively. In the toehold switch, we sent the designed sequences to the IDT Company, and had the toehold switch synthesized with a T7 promoter (BBa_R0085) upstream to it. In order to quantify our detection, we assemble the toehold switch and reporter protein, luciferase. The T7-toehold part is digested with EcoRI and SpeI, then ligated to the luciferase (BBa_I712019) provided by iGEM, which is digested with EcoRI and XbaI. The T7-toehold switch-luciferase is later subcloned to pSB1C3 backbone. As for the trigger part, we construct it to validate the function of toehold switch. The designed trigger is synthesized by the IDT Company, and digested with XbaI and PstI. It then ligate downstream to the T7 promoter (BBa_R0085) digested by SpeI and PstI.Plasmid construction we have done
We construct toehold switch part in pSB1C3 backbone, and construct trigger in pSB1AK8 backbone for use of different antibiotic to check whether the bacteria uptake both plasmids in it.
RESULTS
These two plasmids are what we have constructed. One is toehold switch with T7 promoter and luciferase in pSB1C3 backbone; the other is trigger RNA with T7 promoter in pSB1AK8 backbone. The pSB1C3 T7-toehold-luciferase construct also contains chloramphenicol antibiotic for selection. On the other hand, the pSB1C3 T7-trigger construct contain chloramphenicol antibiotic for selection. The two plasmids are constructed in different backbones for further cotransformation selection, this way we can check whether the two plasmids are both successfully uptaken by the bacteria or not.
Restriction enzyme digestion
(A) 1. Luciferase cut with EcoRI and XbaI.
Lane 1 and 4, 3 μg of luciferase digested by EcoRI and XbaI ; Lane 2, 1K DNA marker ; Lane 3, 3 μg of luciferase not digested by EcoRI and XbaI.
(B) Restriction enzyme digestion of synthetic toehold and trigger RNA with EcoRI and SpeI. Samples were run in 1% agarose gel. Lane 1, 1kb DNA marker；Lane 2-5, 125 ng of synthetic DNA fragment that would transcribe into toehold sensors (SAT、DUSP1、IL-1β、IL-8 ) were digested by EcoRI and SpeI；Lane 6-9, 125 ng of DNA fragment that would transcribe into trigger RNAs (SAT、DUSP1、IL-1β、IL-8 ) were digested by EcoRI and SpeI.
(C) T7 promoter digestion with restriction enzyme PstI and SpeI. Lane 1, marker ; Lane 2, 3 μg T7 promoter digested by restriction enzyme PstI and SpeI.
RESULTS
Preparation of parts to construct two vectors system
We use enzyme to digest, and cut out the bands we want, and then run electrophoresis then amplifying the toehold and trigger RNA we would use. In Figure A, the backbone, pSB1AK8, is 3426 base pairs, when it be digested with restriction enzyme and added luciferase, which is 1653 base pairs, it would be 4937 base pairs. Then we use enzyme EcoRI and XbaI to digest the plasmid. The results match our expectations.
According to Figure B, toehold and trigger RNA. To cut out the toehold and trigger RNAs, we digest them with EcoRI and SpeI. The toehold switch sensor are all 167 base pairs, the trigger RNAs are 149 base pairs. The results match our expectation. In Figure C, we use restriction enzyme PstI and SpeI to digest. Then cut out the T7 promoter, thus further we could amplify the T7 promoter. The T7 promoter part in pSB1AK8 is about 3K .
After we confirm the sequences are correct, we digest toehold switches, luciferases from pSB1AK8, trigger RNAs, and T7 promoters from pSB1C3. Then we construct two vectors, one contains T7 promoter luciferase and toehold switch sequence in pSB1C3 backbone ; the other vector, pSB1AK8, contains T7 promoter trigger RNAs sequence. After construct these two vectors, we sent it to the biotechnology company to confirm the sequence. The confirmed final plasmids are showed in Figure 7. In the future, we will cotransform these two vector into BL21*DE3 and test the luciferase’s brightness, then we can lysate the bacteria, which already have these two plasmids, and use ELISA reader to test whether there is light or not. Further to build the sensitivity standard curve.
Discussion
In Figure , the size of cut is about 5 K. Uncut DNA’s structure is supercoiled, it should be below the cut DNA. After discussing with the advisor, we still don’t know the reason why the uncut doesn’t meet our expectation. We predict that the structure of uncut plasmid didn’t fold perfectly, so it didn't run rapidly.
Luciferase brightness test
After construct two plasmids, we want to test the sensor efficiency of the toehold switch structure. So we then transform the two plasmids into BL21 bacteria then break the bacteria to get lysate. We use PCR to make a positive control, a fragment of sequence contains T7 promoter, toehold switch, and luciferase. And our negative control for this part is T7 promoter with luciferase.
RESULTS
The table is the outcome of the luciferase brightness test. In our prediction, if we add the trigger RNA, which simulate the environment of oral cancer patients’ saliva in real world, the toehold switches we design should be triggered, then the hairpin structure would be opened, downstream reporter gene thus can be translated. For our experimental group and positive control, we used T7 promoter with luciferase, so its reporter gene must be expressed. On the other hand, negative control is T7promoter, toehold switch, and luciferase, but without trigger RNA. As you can see, IL8 ,DUSP1,SAT experimental group’s data did not match with our expectations, for that the negative controls are just as twice of the our experimental data.
PCR for T7-luciferase
In the figure, each lane are added with 10 μl of PCR products, we use temperature gradient (50°C, 53°C, 56°C, 59°C, 62°C, 65°C) to test the product of which temperature condition is suitable to be our negative control of in vivo functional assay.
RESULTS
To do the in vivo part of functional assay, we must have control condition. This figure shows the process that we use temperature gradient to choose which temperature is the most appropriate for us to do the experiment of the in vivo part of functional assay. Finally, we choose the result of 53 °C for PCR. Because the product of 53 °C condition is much clearer, also much brighter then other conditions.