When working on our detection system in the lab, we used different cloning strategies for different purposes. For example, Gibson cloning was used to clone the endo-1,3-β-glucanase and dCas9-reporter constructs into a vector backbone. To generate the different guide RNAs we made use of Golden Gate cloning. The entire team put a lot of time and effort into the laboratory work (Notebook), but not all our cloning attempts ended up being successful. However, we were able to achieve positive and good results in the end.
Generation of guide RNA
After using the guide RNA generator from the Peking 2015 team, we verified the constructs by sequencing. RNA was generated with the HiScribe RNA synthesis kit, followed by using a RNA cleanup kit, we were able to produce different guide RNAs. We verified the quality and concentration of the guide RNA using a quBit instrument (Table 1).
Table 1: Concentration of guide RNA measured with quBit.The concentration of four different guide RNAs was measured, concentration ranged between 40 ng/µl to 370 ng/µl.
|Guide RNA||Concentration ng/µl|
The enzyme glucanase is an important part of our detection system, as it catalyzes the hydrolysis of the β-glucan bond in the yeast cell wall, making it easier to lyse the cell and access the DNA. We were not only able to successfully clone the endo-1,3-β-gluanase into the submission vector (Figure 1) and expression vector (Figure 2), but we were also able it induce its expression (Figure 3). Finally, we could also show the glucanase activity in vitro (Figure 4, Figure 5).
Figure 1: Colony PCR of glucanase in submission vector pSB1C3Positive colonies after Two-part Gibson transformation of glucanase g-block (1-15). Glucanase at the size of 1.388 bp (blue line), colony 11 was further used after checking for correct sequence. Ladder Quick-Load 1 kb (N0468S).
Figure 2: Colony PCR of glucanase in expression vector pBADPositive colonies after Two-part Gibson transformation of glucanase (1-9). Glucanase at the size of 1.388 bp plus overhangs (blue line). Ladder Quick-Load 1 kb (N0468S).
The glucanase-pBAD construct was further used for protein expression. Protein expression was induced by adding arabinose (protocols).
Induction of protein expression
The induction of protein expression was verified after small-/or medium-scale cell culturing, followed by separation of proteins on a 12 % SDS-PAGE (Figure 3). The results show that the endo-1,3-β -glucanase expression was induced after addition of 0.1 % arabinose followed by a 3 h incubation at 30oC (protocol).
Figure 3: SDS-PAGE 12 %Uninduced (1) and induced samples (2&3) loaded on SDS-PAGE. Glucanase at the size of 52 kDa (red arrow).
Screening for Polysaccharide Endo-Hydrolases using Insoluble Dyed Polysaccharide
Insoluble dyed substrate is used to detect enzyme activity (protocol).
Figure 4: 96-well plate assay to test enzyme activity (Megazyme) Absorbance of the glucanase was measured against diluted samples of a standard for enzyme activity (Zymolyase, Nordic Biosite). The glucanase activity was tested with samples from induced cell cultures (crude extract, supernatant and cell pellet). Absorbance was measured 1 h after loading the samples on the 96-well plate.
From Figure 4, it can be seen that the glucanase had an activity in between a 100x and 1.000x dilution of Zymolyase. Of the different cell samples from the induced cell culture, the glucanase from the supernatant had the highest enzymatic activity. We expect the glucanase to have a higher enzymatic activity after further experiments purifying the protein.
Figure 5: Plate assay to display enzyme activity (Glycospot) Three replicates of assay. Upper left corner ‘crude cell culture’, middle left ‘supernatant’, lower left ‘positive control’ (commercially available enzyme mix), upper right ‘crude diluted’, middle right ‘pellet’, lower right ‘negative control’ (empty pBAD vector). All samples on the left parts of the plates show enzyme activity in all replicates.
With our results we showed that our BioBrick, the endo- 1,3-β-glucanase, has enzymatic activity and is able to cleave 1,3-β-glucan bonds. The activity is comparable to a 100x-1000x dilution of Zymolyase. Combined with either mechanical force or a detergent, we expect to be able to selectively lyse yeast cell walls.
Experiments that did not succeed yet
Affinity chromatography of glucanase using FPLC (Fast Protein Liquid Chromatography) We performed different FPLC (Picture 1-4) with different outcomes. The enzyme was not only found in the expected fractions but also in the flow through.
Picture 1-4: Absorption spectra from the FPLC of glucanase. Dark green ‘conductivity’, black ‘imidazole buffer’, light green ‘absorption at 280 nm’, pink ‘absorption at 260 nm’, blue ‘absorption at 214 nm’. Proteins have an expected absorption at 280 nm.
In Picture 1 the glucanase was in fraction A16-21, however, there is also a lot of glucanase present in the flowthrough. In Picture 2 the glucanase is present in fraction A8-10, but again a lot of glucanase was lost in the previous fraction.
In Picture 3 and 4 all the glucanase was released in the flowthrough. We hypothesize that this is due to the column being broken down by the enzymatic activity of the glucanase.
- Although we were not successful cloning the dCas9-beta-lactamase into the submission or expression vector, we went through several different cloning attempts, troubleshooting and optimization strategies.
- For cloning we attempted Gibson assembly, 3A assembly and SOE-PCR. We tried to optimize the protocols using different ratios of substrate, incubation times and gradient PCR.
Future plans for the project
- Continue trying the clone the dCas9-beta-lactamase in the expression vector for expression and purification
- Test if the guide RNAs-dCas9 construct can detect C. albicans DNA
- Purify the glucanase to obtain a purer product.
- Test the glucanase on C. albicans
- Develop different guide RNAs to detect a range of pathogens