Team:ZJU-China/Results
Detect HPV with menstrual blood
Although researches have already proved that menstrual is a suitable sample for HPV detection,1-4 it does not mean menstrual blood can do the same in our project because existing researches are all using PCR and DNA prob to detect HPV, which is totally different with our design. However, for safety reason, we decided not to use menstrual blood sample from HPV infected person. Instead, we did a series of experiments to show menstrual blood will work well in our project.
Pad's blood absorption experiment
The first thing is to make sure how much menstrual blood do we need in one test. According to existing researches, a piece of pad full of blood with 1 cm2 area is surely enough for the HPV examination.1-4 Based on it, we explored how much blood can a piece of 1cm2 pad absorb to know how much menstrual blood we need.
We used water mixed with glycerol to simulate blood and carried out this experiment.
First of all, we cut the middle part of three kinds of pad (A, B and C) provided by Hangzhou Xiaojiemei Sanitary Articles Co., Ltd. The cut part was a rectangle with a length of 5cm and a width of 3cm (Figure 1) (the area is 15cm2). Each kind of product was tested with six samples. Then immersed the cut rectangle into the beaker filled with water-glycerol mix until it fully absorbed the mix and sank to the bottom of the beaker. Finally, clamping a small corner of the rectangle with tweezers and lifting it out of the water surface. When there was no water drop, clamping it out of the beaker. The amount of water reduced in the beaker after the experiment was the amount of water absorbed by the rectangle.
Figure 1. The rectangle cutting, sample collection and process of Pad’s blood absorption experiment.
The results are shown in Table 1. The amount of water that a rectangle can fully absorb is about 4.89789g. It means the amount of water that 1 cm2 area of pad can fully absorb is about 0.326526g, which is converted into 326.526ul, so we need about 330ul of menstrual blood for our detection.
Table 1. Data on the amount of water that can be absorbed by pad with an area of 15 cm2.
Cell lysis experiments
HPV can infect different kinds of epithelial cell, and its genome will stay in host cell nucleus in an episomal form, that is the productive infection. However, in most cases of high-grade cervical intraepithelial neoplasia (HGCIN) and invasive disease, HPV integrated into host’s genome and entered transforming infection phase.5 Thus, although patients’ menstrual blood contains some free virus, we will be able to obtain more HPV genome from the sample if we lysed the cervical epithelial cells in menstrual blood.
We than explored how much water can lyse a cell in PBS. We lysed cells with ddH2O, centrifugated mildly, took supernatant and checked if cells were lysed by detecting if supernatant contains human genome. Primers that amplify the human ABO gene were used to detect human genome by PCR, here is the result:
Figure 2. Result for Cell lysis experiment.
CRISPR/Cas12a Biosensing System
RPA
HPV was divided into different subgroups according to their L1 sequence (see background), thus, we ordered a 500bp fragment each from HPV16 and 18 L1 sequence (Figure 3 and 4) based on Doudna’s work and our sequence alignment.6 Here in this method, we used RPA technology to amplify a part of DNA fragment from L1 sequence (about 150 bp).
Figure 3. Part of 500bp from HPV16 L1 gene. Shows the primers we designed and targets (which is complementary with crRNA) for Cas12a to recognize, PAM is necessary for Cas12a to recognize the target, for FnCas12a & LbCas12a, PAM is TTTN; for AsCas12a, TTN.
Figure 4. 500bp from HPV18 L1 gene.
Firstly, we used PCR to find out the most specific primer pairs. PCR shows a better specificity than RPA, and the higher the annealing temperature is, the more specific the PCR would be. Because of it, we chose low annealing temperature so that we can judge the specificity of the primers easier. In the experiments, we used different primer pairs to amplify HPV16 and 18 L1 sequences separately, 1R did not involve in for its bad specificity according to pre-experiment. Here is our result:
Figure 5. Electrophoresis results of Primer Selection Experiment with PCR. Primers are all designed for HPV16 L1, but used to amplify both HPV16 (up) and 18 (down).
According to the results above, we selected 5 primer pairs which show best specificity in the experiment. They are 1F-4R, 3F-4R and 4F-2R, 3R, 4R. Here, the RPA reaction was incubated at 25 ℃ for 40min in order to make the result clearer.
Figure 6. Electrophoresis results of Primer Selection Experiment with RPA.
Based on the Primer Selection Experiment, we finally decided to use 3F-4R primer pair for further experiments.
DNA cleavage assays with Cas12a
DNA reporter cleavage assay. Firstly, we used PCR products to make sure that Cas12a is still active. Here, all the crRNAs we used are targeted to HPV16 (see Figure 3, Target1-3), for control group (the second lane), the crRNA was replaced by equal volume DEPC water. crRNAs were named by the targets they aimed at, abbreviated as Tg1-3.
Figure 7. DNA reporter cleavage assay using PCR products and a 47bp reporter.
After the activity of Cas12a was ensured and the most specific primer pair was determined, we do the Best Ion Concentration experiment to explore the optimum ions concentration for Cas12a considering that RPA reaction mix has already contain some ions. Results are shown in Figure 7. The experiment was done with FnCas12a and Tg1 crRNA because they showed best DNase activity in pre-experiment. In control group, crRNA was replaced by equal volume of ddH2O. Attention, the reaction was incubated for 60min rather than 80min in this experiment to make sure the difference is obvious enough.
Figure 8. DNA reporter cleavage assay using PCR products and a 47bp reporter.
As the optimum ion concentration was determined by the experiment above, a reaction system with 7.6μL RPA products and 0.4μL 10× reaction buffer will be used in all the experiment following.
The next step we have made was to find out the best Cas12a protein from AsCas12a, LbCas12a and FnCas12a as well as find out the best target in HPV16 L1 sequence. crRNAs are all targeted to HPV16 (Figure 3), thus could not recognize HPV18, which comply with our result.
Figure 9. The comparison between different Cas12a and different crRNA targeting different sequences, shows that crRNA targeting HPV16 could not recognize HPV18 L1 sequence.
The results showed that Fn and LbCas12a are much more activated than AsCas12a, and the order of the Cas12a’s activity with different crRNAs are Tg1 > Tg2 > Tg3. Thus, Tg1 is used for the next experiment.
Fluorophore quencher (FQ)-labelled reporter assay. Now that we have determined the Cas12a and its target to use, we continued our experiment to the next phase: Using FQ-labelled reporter to see if the result would be visible under UV light. Tg1 is used in this experiment for every experimental group.
Figure 10. Result of Fluorophore quencher (FQ)-labelled reporter assay. If the reporter was cut, FAM would be able to emit green florescence, otherwise the florescence would stay red (from TAMRA)
We than replaced UV light with a UV flashlight, and the detection is carried out in lab with daylight lamp turned on rather than in a dark room. Luckily, the florescence is still visible and the result is apparent.
Figure 11. Result of Fluorophore quencher (FQ)-labelled reporter assay. A UV flashlight was used.
Apparently, FnCas12a showed much more higher DNase activity compared with As and LbCas12a.
Until now, we have proved that Cas12a biosensing system is capable for HPV detection and typing. But for the intention to expand the scope of application of PaDetector, we also want to develop quantified detection in addition to qualified detection. Thus, we asked dry lab for help to develop a Real-Time Fluorescence Detection Device. With this device, we compared FnCas12a with LbCas12a as well as Tg1 with Tg2 quantitively and got more convincing results.
Firstly, we compare LbCas12a with FnCas12a quantitatively by using Real-Time Fluorescence Detection Device.
Figure 12. Quantitative comparison between FnCas12a and LbCas12a.
As FnCas12a showed a significantly better activity, we used FnCas12a for further exploration.
We then ensured the specificity of FnCas12a with Tg1 and 2. The results were totally consistent with the results of qualitative experiments that the specificity is very good.
Figure 13. Using Real-Time Fluorescence Detection Device to ensure the specificity of FnCas12a with Tg2 (A) and Tg1 (B).
Finally, we compared the reaction speed when using either Tg1 or Tg2.
Figure 14. Quantitatively compared FnCas12a’s activity when using Tg1 or 2.
Hybridization Chain Reaction Biosensing System
Version 1.0
Characterization by step
Restriction enzyme sites and the target sequences we chose in HPV detection
HPV was divided into different subgroups according to their L1 sequence (see Design), thus, we ordered a 500bp fragment each from HPV16 and 18 L1 sequence (Figure 3 and 4) based on Doudna’s work and our sequence alignment.6 In the hybridization chain reaction biosensing system, we chose EcoRI and PstI to digest HPV16 L1, and BcuI and PstI to digest HPV18 L1. According to the position of selected enzyme cutting sites, we chose 4 sequences to be the target of HPV16 detection and 4 sequences to be the target of HPV18 detection, then designed P2 according to these sequence, which we call P216-1, P216-2, P216-3, P216-4 and P218-1, P218-2, P218-3, P218-4 (Figure 15).
Figure 15. Restriction enzyme sites and the target sequence we chose in HPV detection. A. We chose EcoRI and PstI to digest HPV16 L1. According to the position of selected enzyme cutting sites, we chose 4 fragments to be the target for HPV16 detection, and designed P2 according to these sequences, which we call P216-1, P216-2, P216-3, P216-4. B. We chose BcuI and PstI to digest HPV18 L1. According to the position of selected enzyme cutting sites, we chose 4 sequences to be the target of HPV18 detection, and designed P2 according to these sequences, which we call P218-1, P218-2, P218-3, P218-4.
Pre-processing of HPV genome
Using two kinds of endonucleases combined with ExoIII to digest L1 region from HPV genome (Figure 16). We synthesized 500bp of L1 region from HPV genome and inserted it into pUC19 for detection. For the picture above, 100ng HPV L1 was digested by 0.5μL of each endonuclease and 2U ExoIII in the total volume of 20μL for 4h at room temperature. For the picture below, firstly 100ng HPV L1 was digested by 0.5μL of each endonuclease in the total volume of 20μL for 4h at room temperature, then added 2U ExoIII and digested for 1h at room temperature. Theoretically, HPV16 L1 digested by EcoRI and PstI and HPV18 L1 digested by BcuI and PstI would be the best choice. But as shown in Figure 16, HPV16 L1 digested by EcoRI and PstI almost being completely degraded (above), while HPV16 L1 digested by PstI only is not completely degraded. Considering this, we chose to digest HPV16 L1 by PstI and HPV18 L1 to be digested by BcuI and PstI.
Figure 16. Using two kinds of endonucleases combined with ExoIII to digest L1 region of HPV genome. We synthesized 500bp of L1 region of HPV genome and inserted it into pUC19 for detection. (1) HPV16 L1; (2) HPV16 L1 digested by EcoR1; (3) HPV16 L1 digested by Pst1; (4) HPV16 L1 digested by EcoR1 and Pst1; (5) HPV16 L1 digested by ExoIII; (6) HPV16 L1 digested by EcoR1 and ExoIII; (7) HPV16 L1 digested by Pst1 and ExoIII; (8) HPV16 L1 digested by EcoR1, Pst1 and ExoIII; (9) DL2000 plus DNA ladder; (10) HPV18 L1; (11) HPV18 L1 digested by Bcu1; (12) HPV18 L1 digested by Pst1; (13) HPV18 L1 digested by EcoR1 and Pst1; (14) HPV18 L1 digested by ExoIII; (15) HPV18 L1 digested by Bcu1 and ExoIII; (16) HPV16 L1 digested by Pst1 and ExoIII; (17) HPV16 L1 digested by Bcu1, Pst1 and ExoIII. For the picture above, 100ng HPV L1 is digested by 0.5μL of each endonuclease and 2U ExoIII in the total volume of 20μL for 4h at room temperature. For the picture below, firstly 100ng HPV L1 is digested by 0.5μL of each endonuclease in the total volume of 20μL for 4h at room temperature, then add 2U ExoIII and digest for 1h at room temperature.
ExoIII assisted signal amplification-Version 1.0
It is generally assumed that ExoIII can only cut from recessed or blunt 3’-end of dsDNA, but in our experiment, we found that ExoIII also has the capability to degrade single-stranded DNA (Figure 17), thus digest almost all our probes. ExoIII’s activity to degrade ssDNA has also been mentioned in some literatures.7 Meanwhile, many literatures also mentioned that Exo III cannot digest the thiophosphorylation modified site,8 so we made some of our probe thiophosphorylation modified in order to prevent the nonspecific digestion by Exo III.
Figure 18. ExoIII assisted signal amplification-Version 1.0.(1) DL500 DNA ladder; (2) P20; (3) S10; (4) P20+ExoIII; (5) P20+ExoIII+S10. All of the samples react for an hour at room temperature. The concentration of P20 in lane 2, 4-7 is 1μM. The concentration of ExoIII in lane 4-7 is 1U/μL. The concentration of S10 in lane 5-7 is 0.5μM, 0.25μM and 0.05μM. The reaction system was incubated at room temperature for 1h. Compared with lane 4, when S10 exists, the band of P20 undertook a significant migration.
Hybridization Chain Reaction
Characterization of the HCR of pure hairpin DNA strands is as shown below (Figure 19). Without the initiator (S2), the hybridization chain reaction will not proceed. When initiator (S2) exists, the hybridization chain reaction would then begin. The length of nicked double helix varies with the concentration of initiator (S2).
Figure 19. Characterization of the HCR of pure hairpin DNA strands by 8% and 5% native PAGE. (1) H1; (2) h2; (3) H1+h2; (4−7) H1+h2+S2; (8) DL2000 plus DNA ladder. The concentration of H1 and h2. The concentration of H1 and h2 in lane 1, 2, 3 is 1μM, while in lane 4-7 is 2μM. The concentrations of S2 in lane 4-7 is 200nM, 100nM, 20nM, 10nM respectively. The reaction system was incubated at room temperature for 5h.
We also found it’s important for HCR system to have an optimum ion strength (Figure 20). Without NEBuffer1 in the system, the effect of HCR is obviously worse.
Figure 20. Characterization of the influence of ion strength to HCR by 8% native PAGE. (1) H1; (2) h2; (3) H1+h2; (4-7) H1+h2+S2; (8) DL2000 plus DNA ladder. The concentration of H1 and h2. The concentration of H1 and h2 in lane 1, 2, 3 is 1μM, while in lane 4-7 is 2μM. The concentrations of S2 in lane 4 and 5 is 200nM, and in lane 6 and 7 is 100nM. The reaction mix was incubated at room temperature for 5h.
Lateral flow nucleic acid biosensor
The S2-FITC triggered HCR production and S2-Cy3 triggered HCR production are detected on two kinds of test paper we ordered from a company (Figure 21). On Cy3/FITC test paper, the anti-Cy3 Ab test line is ahead of the anti-FITC Ab test line; While on FITC/Cy3 test paper, the anti-Cy3 Ab test line is behind the anti-FITC Ab test line. Comparing these two kinds of test paper, the FITC/Cy3 test paper shows a higher false positive relatively. For the Cy3/FITC test paper, when testing S2-FITC triggered HCR production, it shows the correct result, when testing S2-Cy3 triggered HCR production, it shows false positive too, but the false positive is lower than that on FITC/Cy3 test paper. We assume that it’s because anti-FITC Ab has poor specificity, so we chose the Cy3/FITC test paper for further detection. What’s more, the color of test line and control line of the test paper we ordered from the company is much lighter than normal test papers.
Figure 21. Detection of HCR production on test paper. The S2-FITC triggered HCR production and S2-Cy3 triggered HCR production are detected on two kinds of test paper. On Cy3/FITC test paper, the anti-Cy3 Ab test line is ahead of the anti-FITC Ab test line; On FITC/Cy3 test paper, the anti-Cy3 Ab test line is behind the anti-FITC Ab test line. The FITC/Cy3 test paper we ordered from the company shows a higher false positive relatively. For the Cy3/FITC test paper, when testing S2-FITC triggered HCR production, it shows the correct result, when testing S2-Cy3 triggered HCR production, it shows false positive too, but the false positive is lower than that on FITC/Cy3 test paper.
We then characterize the Cy3/FITC test paper we ordered from a company by HCR productions with different concentration of S2, aim to see the detection limit of S2 on this test paper. As shown in Figure 22, the detection limit of the concentration of S2 in HCR system is about 100nM, which indicates the test paper we ordered from a company is not quite sensitive.
Figure 22. Characterization of the Cy3/FITC test paper.(1-3) The S2-FITC triggered HCR production’s detection on Cy3/FITC test paper. (4-6) The S2-Cy3 triggered HCR production’s detection on Cy3/FITC test paper. The concentration of S2 in HCR system 1-3 and 4-6 is 500nM, 100nM, 20nM, respectively.
Overall characterization
Combination of ExoIII assisted signal amplification and hybridization chain reaction.
Firstly, we used S10 and P20 which is independent of the target sequence of HPV L1 and P216-1-P218-4, to characterize the combination of ExoIII assisted signal amplification and hybridization chain reaction (Figure 23). Compared with lane 7, lane 8 is darker, which indicates when S10 exists, P20 can be digest by ExoIII and initiate hybridization chain reaction. However, false positives were also shown to some degree, reflecting P20 can also initiate hybridization chain reaction combined with ExoIII without S10.
Figure 23. Combination of ExoIII assisted signal amplification and hybridization chain reaction. (1) P20; (2) S10; (3) H1; (4) h2; (5) H1+h2; (6) P20+H1H2; (7) P20+H1H2+ExoIII; (8-10) P20+H1H2+ExoIII+S10; (11) DL2000 plus DNA ladder. The concentration of H1 and h2 in lane 4-11 is 1μM. The concentrations of P20 in lane 6-10 is 500nM. The concentrations of ExoIII in lane 7-10 is 500nM. The reaction system was incubated at room temperature for 4h.
Combination of ExoIII assisted signal amplification and hybridization chain reaction.
In this part, we mix HPV16 L1 or HPV18 L1 plasmid, the endonucleases we chose, P2 designed for different target sequence of HPV16 L1 and HPV18 L1, H1H2 and ExoIII together to characterize our whole project. The concentration of HPV L1 is 1ng/μL, while P2 is 1μM, ExoIII is 2U/μL, and 0.5μL endonucleases in a total volume of 20μL. The reaction system was incubated at room temperature for 4h. As shown in Figure 24, P216-2, P216-3, P216-4, P218-2 seem to have the potential to detect HPV L1, but it is still not very specific.
Figure 24. Combination of preprocessing of HPV genome, ExoIII assisted signal amplification and hybridization chain reaction. A. (1-4) HPV16 L1 digested by PstI, with P2 designed for different target sequence of HPV16 L1. (5-8) HPV16 L1 digested by EcoRI and PstI, with P2 designed for different target sequence of HPV16 L1. (9-12) HPV18 L1 digested by BcuI and PstI, with P2 designed for different target sequence of HPV18 L1. The reaction system was incubated at room temperature for 4h. B. (1-4) HPV16 L1 digested by PstI, with P2 designed for different target sequence of HPV18 L1. (5-8) HPV16 L1 digested by EcoRI and PstI, with P2 designed for different target sequence of HPV18 L1. (9-12) HPV18 L1 digested by BcuI and PstI, with P2 designed for different target sequence of HPV16 L1. The reaction system was incubated at room temperature for 4h.
Combination of ExoIII assisted signal amplification and hybridization chain reaction.
Test the reaction systems in Figure 24 (Figure 24A 2, 3, 6, 7, 10) on test paper. All samples cannot show a test line (Figure 25). We assume that it is because of the low sensitivity of the test paper we ordered from a company.
Figure 25. Combination of preprocessing of HPV genome, ExoIII assisted signal amplification, hybridization chain reaction and lateral flow nucleic acid biosensor.Test part of the reaction systems in Fig on test paper. All samples cannot show a test line.
Version 2.0
Characterization by step
ExoIII assisted signal amplification-Version 2.0
In version 2.0, we try to replace the signal amplification process with index increase in order to make our detection more sensitive. We refer to an ExoIII-assisted cascade signal amplification strategy for label-free and ultrasensitive chemiluminescence detection method raised by Gao Y et.al in 2014.9
We find ExoIII also has the potential to degrade single-stranded DNA (Figure 26), then digest almost all our probes, and ExoIII’s activity to degrade ssDNA also has been mentioned in some literatures.7 Meanwhile, many literatures also mentioned that Exo III cannot digest the thiophosphorylation modified site,8 so we make some of our probe thiophosphorylation modified in order to prevent the nonspecific digestion by Exo III.
Figure 26. ExoIII’s nonspecific degradation of probes without thiophosphorylation modification. (1) P1 without thiophosphorylation modification; (2) S2 without thiophosphorylation modification; (3) P1-S2; (4) S1; (5) P1-S2 without thiophosphorylation modification + ExoIII; (6-8) P1-S2 without thiophosphorylation modification + ExoIII+S1. The concentration of P1-S2 without thiophosphorylation modification in lane 3-8 is 1μM. The concentration of ExoIII in lane 5-8 is 1U/μL. The reaction system was incubated at room temperature for 1h. ExoIII has the nonspecific activity to degrade the probe without thiophosphorylation modification.
To characterize the ExoIII assisted signal amplification-Version 2.0, we ed a S1 and a P1-S2 independent of the target sequence of HPV L1. Using the thiophosphorylation modified probe, as shown in Figure 27, compared with lane 4, when S1 exists, he band of P1-S2 fail to undertook a significant migration, which indicates P1-S2 is probably not be degraded by ExoIII and release S2.
Figure 27. ExoIII assisted signal amplification-Version 2.0. (1) P1; (2) S2; (3) P1-S2; (4) S1; (5) P1-S2 + ExoIII; (6) P1-S2 + ExoIII+S1. The concentration of P1-S2 in lane 3-8 is 1μM. The concentration of ExoIII in lane 5-8 is 1U/μL. The concentration of S1 in lane 6-8 is 250nM, 100nM, 10nM. The reaction system was incubated at room temperature for 1h. Compared with lane 5, when S1 exists, the band of P1-S2 fail to undertake a significant migration.
Overall characterization
Combine ExoIII assisted signal amplification-Version 2.0 with hybridization chain reaction.
We use we S1 and P1-S2 which is independent of the target sequence of HPV L1 to characterize the combination of ExoIII assisted signal amplification-version 2.0 and hybridization chain reaction (Figure 28). It shows strong false positives, reflecting that P1-S2 can initiate hybridization chain reaction without S1 (Figure 28A, 5)
Figure 28. Combination of ExoIII assisted signal amplification and hybridization chain reaction. A. (1)H; (2) h2; (3) H1+h2; (4) H1+h2+S2; (5) P1-S2+H1H2; The concentration of H1 and h2 in lane 4-5 is 1μM. The concentrations of P1-S2 in lane 5 is 500nM. The reaction system was incubated at room temperature for 4h. B. (1-4) (5-8) P1-S2+H1H2+ExoIII+S1 with different concentration of S1 and ExoIII. The concentration of H1 and h2 in lane 1-8 is 1μM. The concentrations of P1-S2 in lane 1-8 is 500nM. The concentrations of ExoIII in lane 1-4 is 1U/μL, while in lane 5-8 is 5U/μL. The reaction system was incubated at room temperature for 4h.
Version 2.0
In hybridization chain reaction biosensing system - version 1.0, the characterization results are relatively complete. For overall characterization, our system shows the ability to detect HPV L1 sequence but is still not specific enough. By changing the target sequence in HPV L1, we can improve the specificity of our biosensing system. Meanwhile, the test paper we ordered from a company seem to have some problem, the anti-FITC antibody is not specific enough, the color of test line and control line of the test paper is also much lighter than normal test papers, so by improve the quality of test paper we can also lower our detection limit and increase the sensitivity.
In hybridization chain reaction biosensing system - version 2.0, theoretically, the new ExoIII assisted signal amplification method should produces an exponential signal amplification, the literature of Gao, Y9 also shows a nice result, but in our experiment, we found it shows strong false positive.
Take all of the above into consideration, hybridization chain reaction biosensing system - version 1.0 is a better and more complete way of HPV detecting.
Cas12a Expression and Purification
We obtained AsCas12a and LbCas12a fragments from purchased plasmids by PCR and linearized pET-28a(+) plasmid by PCR. PCR are also introduced HindⅢ and EcoR1 enzyme cutting sites, so we linked AsCas12a and LbCas12a fragments and pET-28a(+) plasmid to AbCas12a and LbCas12a expression plasmids with His tag by T7 ligase respectively. Agarose gel electrophoresis was used to purify AsCas12a and LbCas12a fragments and linearized pET-28a(+) plasmid (Figure 29).
Figure 29. AsCas12a fragment, LbCas12a fragment and linearized pET-28a(+) plasmid.
We then transfer recombinant plasmids into E. coli BL21 and test them by colony PCR (Figure 30).
Figure 30. Colony PCR results of AsCas12a and LbCas12a.
Cas12a Purification
We cultured recombinant BL21 strains to an OD600 nm of 0.6-0.8 at 200rpm at 37 ℃. Then we induced proteins expression with 0.13mM IPTG and cultured them at 120 rpm at 18 ℃ for 12-16 hours10. After that, we broke up bacterias to get protein supernatants with 10mM imidazole binding buffer. Finally, we purified AsCas12a and LbCas12a proteins using Ni-NTA Sefinose Resin with 10mM imidazole wash buffer and 200mM imidazole elution buffer. SDS-PAGE (Figure 31) and western blot (Figure 32) was used to test protein purification.
Figure 31. SDS-PAGE results of AsCas12a and LbCas12a.
Figure 32. Western blot results of AsCas12a and LbCas12a with Mouse-anti-His antibody.
Besides, we determined the protein concentrations with BCA kit.
Figure 33. BSA standard curve.
Table 2. Protein concentration
Finally, we tested the activity of AsCas12a and LbCas12a by detecting the fluorescent. The extracted AsCas12a and LbCas12a had ability to cut DNA compared with the control group and the extracted AsCas12a was significantly better than the bought AsCas12a (Figure 34).
Figure 34. AsCas12a and LbCas12a activity assay.
Supplementary data
Reference
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