Team:TU Eindhoven/Results
Results of our project
Results Paired dCas9-Split-NanoLuc system
To evaluate our paired dCas9-Split-NanoLuc system, test DNA in the form of gBlocks was designed (Figure 1). The sequence of this test DNA was based on the T7 phage genome since the T7 phage is used for the proof-of-concept of dCastect. By using the T7 sequence, the same gRNA pairs can be used for all experiments. Using the gBlocks, we first wanted to optimize the sensor by varying the protein concentrations and interspace distance. Subsequently, the limit of detection could be determined, followed by tests where the sensor proteins bind to multiple binding sites on the same target to see if an even smaller limit of detection could be reached.
Figure 1: Schematic representation of the design of the test DNA gBlocks.
Variable interspace distances
The interspace distance was varied between the two dCas9 domains to find the optimal distance for association of SmallBit and LargeBit. Because of the helical shape of DNA, we expected to see a pattern with higher signals when the two proteins bind on the same side of the helix and lower signals when the two proteins bind on the opposite side of the helix. Eventually for high interspace distances, we expected a lack of signal because the distance would be too big for association of SmallBit and LargeBit. In the first test we tested interspace distances from 10 bp to 30 bp (see Additional bioluminescence measurements dCas9-Split NanoLuc system). In this test it was seen that interspace distance 30 gave the highest signal. Therefore, we decided to design gBlocks to test interspace distances 10 – 60 bp to find the optimal interspace distance (Figure 2). The tests were performed using a 1.2 nM DNA concentration, 2 nM protein concentration and 6x molar excess of gRNA. The results show that the systems containing an interspace distance lower than 20 bp give a low signal. From 20-60 bp the signal is high, which means that interspace distance is not a limitation for choosing desired gRNA sequences. Interspace distances 30, 50 and 60 give the highest signals.
Figure 2: Bioluminescence intensity measured with variable interspace distances. The interspace distance is the number of base pairs between the two gRNA pairs.
However, it can be observed that the different gRNA pairs have an influence on the overall signal (Figure 2). gRNA pair 1 gives a significantly lower signal for all measurements. To test whether this lower signal is caused by the gRNA and not by the difference in interspace distance, the system containing interspace distance 30 was tested using all three different gRNA pairs (Figure 3). As can be seen, gRNA pair 1 gives a significantly lower signal than the other two gRNA pairs. gRNA pair 3 gives the highest signal, so it was decided to use this gRNA pair in future measurements.
Figure 3: The difference in bioluminescent signal for the three different gRNA pairs.
Subsequently, it was decided to design gBlocks to test even higher interspace distances, since interspace distances of 50 and 60 bp still give a high bioluminescent signal. According to our calculations, interspace distances up to 70 bp should give a measurable bioluminescent signal. In the next test, we tested interspace distances from 20 – 150 bp (Figure 4). It can be observed that an interspace distance of 110 bp gives the highest signal. However, we expected that ±70 bp would be the highest interspace distance that could give a signal, taking into account the approximate distance between PAM-site and C-terminus of dCas9 as well as the length of the linker between this C-terminus and Lbit/Sbit of Split-NanoLuc. A possible explanation for the high signal at 110 bp is the persistence length of dsDNA. The persistence length is a material property of a polymer quantifying its stiffness. If the length of a piece of polymer, like DNA, is much longer than its persistence length, it behaves highly flexible due to thermal energy. If significantly shorter however, the polymer is stiff. The persistence length of DNA is between 35 – 50 nm [1, 2, 3], which corresponds to the length of 103-147 base pairs. The 110 bp interspace distance thus matches this persistence length, meaning this long interspace is semi-flexible. Therefore, we expect the target sites could come closer to each other, hence it would be possible for the two dCas9 proteins to find each other even though they do not bind close to each other on the DNA strand.
Figure 4: Bioluminescence intensity with interspace distances from 20 bp to 150 bp.
Variable protein concentrations
Another test was executed to determine which range of protein concentrations would be most reliable for the detection of DNA. We tested protein concentrations 2, 10, 20, 50 and 100 nM with a DNA concentration of 1.2 nM. The results show that a protein concentration of 10 nM has the best signal-to-noise ratio (Figure 5). At higher protein concentrations, an increase in background signal can be observed without a significant increase in signal. For 100 nM, the background signal is as high as the actual signal, indicating that higher protein concentrations do not increase the sensitivity of the sensor.
Figure 5: Bioluminescence intensity when the protein concentration was varied from 2 nM to 100 nM. Background signal containing only protein and gRNA and a control using non-target DNA were also tested.
Variable DNA concentrations
We wanted to test the limit of detection of our sensor. Therefore, we tested a range of DNA concentrations from 1 pM to 10 nM using a protein concentration of 2 nM (Figure 6A) and 10 nM (Figure 6B). It was expected that the 2 nM sensor would be more sensitive than the 10 nM sensor and would therefore be able to detect lower DNA concentrations. It was shown that the 10 nM sensor is better at detecting higher DNA concentrations. The limit of detection then is 50 pM. The 2 nM sensor is indeed more sensitive and can detect DNA concentrations as low as 10 pM. Also, the Hook effect can be observed for both sensors, which is the decrease in signal at greater concentrations of the target compound. Due to the high availability of binding sites, the likelihood of either dCas9-SmallBit or dCas9-LargeBit binding at a binding site is higher. Therefore, SmallBit and LargeBit do not associate and a decrease in bioluminescent signal can be observed.
Figure 6: A) Bioluminescence intensity when the target DNA concentration is varied at a 2 nM protein concentration. B) Bioluminescence intensity when the target DNA concentration is varied at a 10 nM protein concentration.
Multiple sensor proteins
The limit of detection was determined using one dCas9-SmallBit and one dCas9-LargeBit binding to the recognition sites on a target sequence. We wanted to see if the limit of detection could be lowered when multiple sensor proteins would bind to multiple recognition sites on one target DNA strand. We tested the limit of detection using three pairs of sensor proteins on one target (Figure 7). The results show that the limit of detection is slightly lowered to 5 pM when three pairs of sensor proteins bind to multiple recognition sites on one target.
Figure 7: Bioluminescent intensity when three sensor protein pairs bind to three paired recognition sites.
dCastect proof of concept
It was not allowed to work with phages at the Eindhoven University of Technology. Therefore, we went to Brussels and denatured the phages. In Eindhoven we measured the denatured phages with our sensor. This is further explained on the Demonstrate page.
Future outlook
Statistical relevance
First of all, most experiments need to be repeated several times to obtain more statistical relevant results. Within our limited timeframe we have not been able to repeat all our experiments for multiple times to realize statistical relevance and more results are needed to achieve this, however certain trends within our results have been observed and discussed.
Paired dCas9-Split-NanoLuc system
The optimal conditions for the paired dCas9-Split-NanoLuc system have been determined. The next step would be to test with actual patient samples. Patient samples contain a more complex medium with other proteins and DNA present. After validation with patient samples, the detection proteins can be incorporated into a device.
Single dCas9-BRET system
After many trials with classical ligation and Gibson assembly, we were unable to form the DNA construct of our dCas9-BRET fusion protein. We find this very unfortunate, because for now we do not know if our design of the dCas9-BRET fusion protein could be functional. If the design would be functional, it would be a ground-breaking ratiometric detection protein. Hence, research will be continued at our university after the iGEM competition.
dCastect
Our proof of concept was based on the T7 phage. The next step would be to infect bacteria with phages and measure the phage DNA after the replication and heat shock. Subsequently we would validate the system with other phage types so other bacterial strains can be detected. As mentioned in the collaboration with Stockholm, we could use our system to detect which one of their phages is needed to kill a certain bacterium.
Project achievements
Successes
- Additional bioluminescence measurements dCas9-Split NanoLuc system
- Expression dCas9 addgene
- dCas9 replacement and expression of dCas-Split-NanoLuc system
- Cysteine-Free-NanoLuc expression and characterization
- mCherry expression and characterization
Failures: The road to success
- Expression dCas9-Split-NanoLuc system
- Bioluminescence measurements dCas9-Split-NanoLuc system
- Assembly of IDT gBlocks for dCas9-BRET into our vector
References
- Gross, P., et al. (2011). Quantifying how DNA stretches, melts and changes twist under tension. Nature, 7, 731-736.
- Cifra, P., Benková, Z. & Bleha, T. (2010). Persistence length of DNA molecules confined in nanochannels. Phys. Chem. Chem. Phys., 12, 8934-8942.
- Brinkers, S., Dietrich, H.R.C., de Groote, F.H., Young, I.A. & Rieger, B. (2009). The persistence length of double stranded DNA determined using dark field tethered particle motion. J. Chem. Phys, 130(21), 215105.
Expression dCas9 addgene
The sequence of the S. pyogenes derived catalytically inactive Cas9 was a gift from David Liu (Addgene plasmid #62935). After experiencing difficulties with expressing our own dCas9 fusion proteins, expression of this Addgene dCas9 was performed to optimize conditions for later expression of our dCas9 fusion proteins.
The protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). All the purification steps (lysate flow through, bind flow-through, wash flow-through, elution) and a concentrated sample of the elution were put on an SDS-PAGE gel to evaluate the purification process. dCas9 has a molecular weight of 158 kDa. A clear band is visible in the elution around 158 kDa. It is remarkable that there is also a band at this height in the wash flow-through. This indicates that the 30 mM imidazole of the wash buffer already causes the protein to be released from the column. Furthermore, a very big band is visible in the sample of the concentrated elution. To conclude, dCas9 was successfully expressed.
Figure 1: SDS-PAGE gel of Nickle affinity chromatography samples.
dCas9 replacement and expression of dCas-Split-NanoLuc system
dCas9 replacement
The dCas9 sequence of the original vector could not be expressed. Therefore, the dCas9 sequence was replaced by the dCas9 sequence of the Addgene plasmid which was a gift from David Liu (Addgene plasmid #62935). Overhang PCR was used to include the restriction sites needed to replace the dCas9 in the vector. With colony PCR we confirmed that the Addgene dCas9 was ligated into the vector. All successful colonies were transformed into NovaBlue and after small culture miniprepped. The samples with the highest DNA concentration for dCas9-SmallBitNanoLuc and dCas9-LargeBitNanoLuc were transformed and expressed. The complete sequences were confirmed with sequencing.
Figure 1: Colony PCR gel of dCas9-SmallBitNanoLuc.
Figure 2: Colony PCR gel of dCas9-LargeBitNanoLuc.
Expression
The fusion proteins were successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). The elution was collected in 5 fractions of 1.5 mL. The three elution samples with the highest concentrations were put on an SDS-PAGE gel to evaluate the purity and molecular weight of the samples. dCas9-SmallBitNanoLuc has a molecular weight of 162 kDa and dCas9-LargeBitNanoLuc has a molecular weight of 178 kDa. As shown in figure 3, the blobs of dCas9-LargeBitNanoLuc are a bit higher compared to the blobs of dCas9-SmallBitNanoLuc. This was expected when looking at the molecular weights of the two fusion proteins. There are other bands visible, so the purity is not optimal. However, the contrast between the huge blobs and the other bands is very big.
Figure 3: SDS-PAGE gel of IMAC elution samples of dCas9-SmallBitNanoLuc (Elute 2: 20.79 μM, Elute 3: 7.298 μM, Elute 4: 12.08 μM) and dCas9-LargeBitNanoLuc (Elute 2: 17.90 μM, Elute 3: 6.37 μM, Elute 4: 2.88 μM).
Additional bioluminescence measurements dCas9-Split NanoLuc system
Incubation conditions
First, we tested which incubation conditions were most suitable to test the sensor. The incubation of dCas9 with gRNA can best be performed for 20 minutes at 37 °C. The subsequent incubation of the dCas9-gRNA complex with DNA should be done for at least 10 minutes at room temperature. Furthermore, gRNA can be heated for 5-10 minutes before use to prevent the formation of secondary structures. However, the incubation temperature can be optimized. Therefore, we decided to test incubation temperatures of 37 °C and 50 °C (Figure 1). As can be seen, the bioluminescent signal for all three sensors binding to target DNA is higher after gRNA incubation at 50 °C. Also, the bioluminescent signal of both control groups is lower for 50 °C. Therefore, in all bioluminescent experiments, an gRNA incubation condition of 50 °C has been used.
Figure 1: Bioluminescent intensity of samples when gRNA is incubated for 5-10 minutes at either 37 °C or 50 °C.
PAM orientation
It was explained that the PAM-In orientation was chosen for the design of our paired dCas9-Split-NanoLuc system. Although this design was based on literature, we still wanted to test if we made the right choice. Thus, we tested the bioluminescent signal using the PAM-Out orientation for interspace distance 25 (Figure 2). The results show that the mean bioluminescent signal for the PAM-Out orientation is much lower than the mean bioluminescent signal for PAM-In. From these results, we derived that PAM-In was a good choice.
Figure 2: Bioluminescent intensity when PAM-Out orientation is used.
Variable interspace distance
We designed the first gBlocks and corresponding gRNA pairs in a way to be able to test interspace distances from 10 to 30 bp (Figure 3). This was based on literature and PyMOL measurements of our sensor. We performed bioluminescent tests to find the optimal interspace distance using a 2 nM protein concentration, a 1.2 nM DNA concentration and a 6x molar excess of gRNA. The results show an optimal interspace distance of 30 bp. As expected, interspace distance 20 also gives a relatively high signal, which is due to the orientation of the helix. The proteins then bind at the same side of the helix, resulting in a higher signal. After this test, it could still be possible that an interspace distance higher than 30 bp would be optimal, since we were not able to test this. Thus, in later tests, higher interspace distances were tested to check whether the optimal interspace distance is indeed 30 bp (see Results Paired dCas9-Split-NanoLuc system).
Figure 3: Bioluminescent intensity when interspace distances from 10 bp to 30 bp were tested.
Limit of detection using lower protein concentrations
We tested the limit of detection for 2 nM and 10 nM sensor protein concentrations (see Results Paired dCas9-Split-NanoLuc system), which was as low as 10 pM for a 2 nM sensor protein concentration. To check if the limit of detection could be lowered, we tested even lower protein concentrations: 1 nM (Figure 4A) and 0.5 nM (Figure 4B). For the 1 nM sensor protein, 5 pM is the lowest DNA concentration that can be distinguished from the background signal. However, the difference in signal with the background signal is only minimal. For 0.5 nM sensor protein, the limit of detection is 10 pM, which is the same as for 2 nM sensor protein. Therefore, the limit of detection cannot be lowered significantly using lower sensor protein concentrations.
Figure 4: The effect of testing a variety of DNA concentrations using A) 1 nM sensor and B) 0.5 nM sensor.
Cysteine-Free-NanoLuc expression and characterization
Cysteine-free-NanoLuc was cloned into a pET28a(+) vector, subsequently expressed in BL21 (DE3) E. coli and purified using Ni-NTA affinity chromatography and Strep-Tactin purification. Afterwards, expression was analyzed on SDS-PAGE (Figure 1). This shows that after the purification there is a clear blob between 20 kDa and 25 kDa which corresponds with the molecular weight of the 6xHis-tagged and Strep-tagged Cysteine-free-NanoLuc, which is 22 kDa.
Figure 1: SDS-PAGE of Cysteine-free-NanoLuc (319 µM) after purification.
Functionality test
After successful expression and purification, measurements could be performed to determine whether cysteine-free-NanoLuc was still functional and could be used as a replacement for NanoLuc in fusion proteins where subsequent maleimide coupling is required. Bioluminescence intensity was measured for different concentrations of both NanoLuc proteins (Figure 2). This shows that cysteine-free-NanoLuc has a similar shape of the emission spectrum and has its maximum at the same wavelength as NanoLuc. It furthermore shows that cysteine-free-NanoLuc is still functional, although showing a decrease of about fifty percent in intensity for all measured concentrations. However, due to NanoLuc’s initial brightness, cysteine-free-NanoLuc is still a lot brighter than most other luciferases while also maintaining its small size and high stability.
Figure 2: Emission spectrum (A) and maximum intensity at 460 nm (B) of Cysteine-free-NanoLuc and NanoLuc.
Expression dCas9-Split-NanoLuc system
The vector ordered at Genscript (pET28a(+)) with our insert was dissolved and transformed into BL21. Three different large cultures were used for protein expression; LB, 2YT and TB. When the OD600 had reached 0.6-0.7, protein expression was initiated by adding 1 mM IPTG. After protein expression, purification was done using the His-tag at the beginning and the Strep-tag at the end of the construct. No protein concentrations were measured with NanoDrop. All the samples from the purification were put on an SDS-PAGE gel. From the results we concluded that the expression had failed. There was no protein present at all, not even in the pellet. Therefore, a new plan was made to test different combinations of expression conditions based on information we gathered. We had contact with the iGEM team Dresden to troubleshoot our problem and we also looked in literature. We suspected that dCas9 might be a bit toxic for the bacteria. Therefore, we wanted to temper protein expression by changing some expression condition. Four different condition combinations were tested, see Table 1. The following conditions stayed the same for all four samples; BL21(DE3) as host, LB as grow medium, expression initiation at OD600 = 0.5. The cultures were lysed with ultrasound, a less aggressive lysis method compared to BugBuster which was used at first.
| IPTG (nM) | Temperature (°C) | |
|---|---|---|
| 1. | 0.2 | 16 |
| 2. | 1.0 | 16 |
| 3. | 0.2 | 20 |
| 4. | 1.0 | 20 |
The expression of our protein was still unsuccessful. After purification, the samples were run on an SDS-PAGE gel. No bands were visible at the expected height. We found out that the sequence of our dCas9 was different compared to the team from Dresden and other sources from literature. While ordering our sequence, codon optimization was applied. Apparently, this was the cause of our expression problem. We ordered a vector from Addgene with the correct sequence and replaced our sequence for dCas9. Now we were able to express our protein! You can find these results here.
Bioluminescence measurements dCas9-Split-NanoLuc system
First tests
The protein was successfully expressed, so the first bioluminescence tests were performed to check the function of our sensor. The tests were performed using 10 nM protein and 1.2 nM DNA. The molar ratio of protein : gRNA was 3:4. Several controls and dCas9 complexes containing interspace distances of 17, 20 and 22 were tested and the substrate furimazine was added in a 1000X dilution to each sample. A PBS buffer containing 0.1% BSA was used for all samples. The bioluminescence was tested using the Tecan Spark 10M Plate Reader in a 384 Wells Plate using 50 μL samples (Figure 1).
Figure 1: First bioluminescence test.
As can be seen in Figure 1, the background samples containing either only dCas9-SmallBit or dCas9-LargeBit give a very low signal. However, the control sample containing both dCas9-SmallBit and dCas9-LargeBit without gRNA and DNA and the control sample containing both proteins and target DNA without gRNA give a high signal.
Variable DNA concentration
In the first tests, the control samples give a high signal. We decided to vary the DNA concentrations while testing the dCas9 complexes with the same interspace distance. The test was performed in triplicate with 50 μL samples. The protein concentration was 10 nM, the molar ratio of protein : gRNA was 3:4 and DNA concentrations of 0.6 nM, 1.2 nM and 2.4 nM were used. Protein and gRNA were incubated for 20 minutes at room temperature and this complex together with DNA was incubated at 37 °C for 30 minutes. The substrate was added in a 1000X dilution to each sample. The same buffer and plate reader were used as in the first test. The results repeatedly show high bioluminescent signal for the control samples (Figure 3). When the DNA concentration is varied, the difference in signal is not substantial (Figure 2). In addition, the controls show a much higher signal than the samples containing target DNA, so these tests are not reliable yet.
Lower protein concentrations
To hopefully reduce the background signal, tests were performed using lower protein concentrations. Concentrations of 1 nM and 0.5 nM were tested, together with DNA concentrations of 1.2 nM and a 3:4 molar ratio of protein : gRNA. Incubation of protein with gRNA was done for 20 minutes at room temperature and the subsequent incubation with DNA was done at 37 °C for 30 minutes. The substrate was added in 2000X dilution. The same buffer and plate reader were used as previously mentioned.
Figure 4: Relative bioluminescence during testing with lower protein concentrations.
The results show that the background containing only the proteins without gRNA and DNA give the highest signal (Figure 4). The samples that contain target DNA give the lowest signal, which is not what we expected.
Variable incubation conditions
We read more about dCas9 in literature and several researchers had problems with aggregation when dCas9 is incubated at 37 °C [1]. Therefore, we decided to vary the incubation conditions to reduce aggregation. In addition, DTT was added to the buffer. Tests with 50 μL sample were performed in duplicate. 10 nM protein, 1.2 nM DNA and a 3:4 molar ratio of protein : gRNA was used. The tests were performed using a 2000X dilution of furimazine in the same plate reader as previously mentioned. Incubation of dCas9-gRNA and DNA was done for 10 minutes and 30 minutes at room temperature and for 30 minutes at 37 °C.
Figure 5: Relative bioluminescence of the samples when incubation conditions vary.
The background signal of the control groups did not decrease under different incubation conditions (Figure 5). It can be seen that the bioluminescent signal of the control groups is lowest in the samples that were incubated at 37 °C, so either no aggregation occurs or aggregation is not prevented under these testing conditions.
Variable interspace distance
In the previous tests, only the samples with interspace distances of 17, 20 and 22 were tested. Our DNA and gRNA combinations make it possible to test samples with interspace distances of 10, 12, 15, 17, 20, 22, 25, 27 and 30. Therefore, we decided to test all possible interspace distances in order to find the optimal distance that would give a signal higher than the background signal. 50 μL samples were tested in duplicate, using the same buffer containing DTT. The same concentrations of protein, DNA, gRNA and furimazine were used as in the previous test. Incubation of dCas9-gRNA and DNA was performed for 30 minutes at room temperature.
The samples with interspace distances 10, 12 and 15 (blue), 17, 20 and 22 (red) and 25, 27 and 30 (green) are measured using the same DNA strand (Figure 7). The samples with interspace 10, 17 and 25; 12, 20 and 27; and 15, 22 and 30 are measured using the same gRNA. A pattern can be observed, since the samples measured with the same gRNA give approximately the same signal. Furthermore, the control samples give a high signal (Figure 6).
Centrifugation
The relative bioluminescent signal for the control samples is still high, and aggregation is the most likely explanation. Therefore, in this test the samples were centrifuged for 5 minutes at 13,400 rpm and the supernatant was used for testing. The same concentrations for protein, DNA, gRNA and furimazine as in the previous tests were used. 50 μL samples were used in the same buffer containing DTT. Incubation of dCas9-gRNA and DNA was performed for 30 minutes at room temperature.
Figure 8: Relative bioluminescence of samples after centrifugation for 5 minutes at 13,400 rpm.
Centrifugation of the samples did not decrease the high background signal of the controls (Figure 8), since a high signal can be observed.
Higher molar excess of gRNA
In several papers about dCas9, a higher molar excess of gRNA is used. We decided to test with a molar excess of 6x, to make sure dCas9 can successfully form a complex with gRNA. 30 μL samples were made, using the same buffer containing DTT. The same concentrations of DNA and furimazine as in previous tests were used. A 2 nM protein concentration and 6x molar excess gRNA was used. Incubation was done for 30 minutes at room temperature.
Figure 9: Relative bioluminescence of the samples when tested with a 6x molar excess of gRNA.
The difference in bioluminescent signal between samples containing different interspace distances is minimal (Figure 9). The control samples give a very high signal, which is about 4x as high as the samples containing target DNA. The control sample containing non-target DNA gives a slightly higher signal than the samples containing target DNA. Therefore, the gRNA excess did not improve the functioning of the sensor.
Testing with sgRNA instead of gRNA
For testing, we ordered tracrRNA and crRNA, which need to be incubated to form guideRNA. To make sure the guideRNA is functional, we ordered complete sgRNA for testing. 30 μL samples were made, using the same PBS buffer containing 0.1% BSA and 5 mM DTT. Some samples were tested without DTT. The same protein, DNA, sgRNA and furimazine concentrations were used as in the previous test. Incubation was done for 30 minutes at 37 °C.
Figure 10: Relative bioluminescence of the samples when tested with sgRNA instead of tracrRNA + crRNA.
The results show that the sgRNA did not make a significant difference (Figure 10). The signal for the controls is still higher than the signal for the samples containing target DNA. However, this difference in signal is much smaller.
Testing with Mg in buffer
It is suspected that we are not measuring a real signal yet in the samples containing target DNA. It is known that magnesium plays an important role in the interaction between proteins and DNA. In the case of Cas9, it can accelerate the binding time of dCas9 to DNA up to 800x when magnesium is present [2]. We made a new buffer containing 20 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, 5% glycerol and 5 mM DTT. 30 μL samples were made, where the same protein, DNA, RNA and furimazine concentrations were used. Incubation was performed for 30 minutes at room temperature.
Figure 11: Relative bioluminescence measured in samples when Mg is used in the buffer.
The relative bioluminescent signal is still high for all control samples, especially for the control sample containing non-target DNA (Figure 11).
Finally, we found the mistake causing us to measure only background signal. In the beginning a mistake had been made with the calculations of the DNA concentrations. This resulted in the addition of 1.2 pM instead of 1.2 nM DNA. We diluted the DNA samples to 1.2 nM and tested again, which resulted in a good signal when target DNA was present.
References
- Sternberg, S.H., LaFrance, B., Kaplan, M. & Doudna, J.A. (2015). Conformational control of DNA target cleavage by CRISPR-Cas9. Nature, 527, 110-114.
- Raper, A.T., Stephenson, A.A. & Suo, Z. (2018). Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9. J. Am. Chem. Soc., 140(8), 2971-2984.
Assembly of IDT gBlocks for dCas9-BRET into our vector
We used the IDT offer to synthesize the dCas9-BRET construct. The construct was so big that it had to be ordered in two gBlocks. Restriction sites were incorporated to ligate the two gBlocks and to ligate the complete construct into our expression vector. At first, we tried to ligate the two insets at once in the vector after digestion. This was unsuccessful. After this we tried it again. Furthermore, we also tried to ligate one insert at the time. This was also unsuccessful. We decided to try Gibson assembly. Our gBlocks were not designed for Gibson assembly, so we performed overhang PCR to incorporate the overhangs for Gibson assembly. However, after Gibson assembly still no colonies were present and a control for the transformation was included. We have tried it again but still no results. From a member of the iGEM team of Stockholm we heard that it is better to use PCR linearization instead of digestion. This was also tried, but still no results. We have designed new gBlocks with the new overhangs and order them at IDT. Due to a lack of time, we were unable to make it work.
