Difference between revisions of "Team:Munich/software"

We mainly developed two branches of Software needed for our project. On the one hand, we developed Software to allow user's devices such as Computers and Smartphones to control our Hardware's devices, Heatbringer and Lightbringer. On the other hand, we used scripting in order to improve the performance of the Cas13a protein regarding a diagnostic device test. This involved the post-design verification of crRNA regarding secondary structure and transcriptomal uniqueness as well as the development of a database of crRNA designs that have already worked. We tried to make the latter as extensive as possible given the limited time, checking for collaboration with other teams working with Cas13a, mainly TU Delft.

crRNA Design Verification

There are two main problems regarding the crRNA design of Cas13a for a diagnostic device. First of all, one needs to make sure that the secondary structure of the crRNA needed for Cas13a activity is achieved. Second, one needs to make sure that the sequence targeted by the crRNA is specific, i.e. there is no off-target effects in the transcriptome of the organisms present in the sample. If this is not the case, false positive results will occur. The software we developed relies mainly on bioinformatic principles such as Secondary Structure Prediction and Basic Local Alignment Searches Tools (BLAST).

Plate.

Cuvette

Mfold

Mfold is a webserver for RNA secondary structure prediction developed by Michael Zuker based on his paper "Mfold web server for nucleic acid folding and hybridization prediction" that published in Nucleic Acids Research in 2003. Since Mfold is not available as a locally buildable binary for every operating system, we developed a script that automatically requests a standardised RNA Fold job to the server, therefore making it available throughout all operating systems. Using the result obtained from this request, the secondary structure is checked via a string comparison in so-called "Vienna" notation. This notation gives base pairing as a string of dots and brackets where a dot represents a non-bonded base and brackets form the base-pairs, clarified by a opening bracket "(" at the 5'-end of the base-pair and a closing bracket ")" at the 3'-end. An example taken from the sample output of the program is given below:
NICE! YOU'VE GOT THE RIGHT SECONDARY STRUCTURE! YOUR SEQUENCE WAS: GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACACUUUACUCCCUUCCUCCCCGCUGAAAGAU (.((((((.((((....)))).)))))).) ######## MATCHED SECONDARY STRUCTURE .....................(.((((((.((((....)))).)))))).).............. ######## PREDICTED SECONDARY STRUCTURE YOUR BACKBONE SEQUENCE HAS BEEN FOUND IN THE DATABANK IT CORRESPONDS TO THE SEQUENCE OF: lwaCas13a

Plate reader configuration:

Gain: 600
Number of flashes per well (absorbance): 22
Number of flashes per well (fluorescence):: 40
Temperature: 37 °C
Filter: Dichroic filter 491.2 nm
Emission wavelength: 515-20 nm
Excitation wavelength: 470-15 nm
Fluorescence reading: bottom optics

Plate reader picture

Results and Discussion:

Device 1 from Plate 7 was problematic to transform. We obtained no colonies after trying several times chemical transformation and electroporation, so we think there was some problem with that specific well. Then, we used the Device 1 from Plate 6, and we obtained several colonies that we could use for the InterLab. Later, we found that in the Registry of Standard Biological Parts website, it is stated that Device 1 is a “complicated sample”, so this could mean that there is some inherent problem with the device itself.

Most devices showed a similar growth behavior except Device 1, which had the lowest growth rate. In terms of fluorescence, the Device 2 produced the highest amount of fluorescence, even more than the positive control, followed by Device 4. The Device 3 and 6 produced no fluorescence. Device 1 and 5 produced very little fluorescence after 6 hours.

Since all the transformed bacteria showed very similar growth patterns across the devices, the lack of fluorescence can be attributed to a problem in the device itself or a transformation problem.

Regarding the plate reader measurements, bubble formation was problematic because it led to wrong measurements or outliers in our data. We solved this by checking for bubbles before measuring and we set the plate reader to lightly shake the plate while measuring the data. Also, we did three measurements per plate to minimize the outliers.

Conclusion

Device 2 showed the best fluorescence results, even better than the positive control. Device 4 was the second one with the highest emission. Device 1 was problematic from the beginning, so probably the low growth rate and low fluorescence emission was caused by a problem with the plasmid sequence. Device 5 also emitted low fluorescence, and device 3 and 6 showed no fluorescence.