DNA

Part:BBa_K3791022

Designed by: Laura Snchez Ruiz, Auba Fuster Pal   Group: iGEM21_UPF_Barcelona   (2021-10-12)


Efficient gRNA Erythromycin construct

This composite part contains an efficient gRNA for CRISPRS-Cas12 use and the necessary elements to allow for the proper transcription of the erythromycin efficient gRNA (BBa_K3791017): promoter and terminator. In contrast to the constructs for the transcription of the preliminary gRNAs (not efficient), apart from containing the efficient gRNAs, a terminator was added in order to ensure proper transcription ending. In this way, the gRNA performance improves, thus obtaining better results with this construct when compared to the not efficient (initial) ones.

The promoter used was T7 (BBa_K1614000), which is a strong promoter that allows transcribing a high amount of the gene of interest in a short period of time. When it is used in pertinent competent cells, it is indirectly inducible by the addition of IPTG (as the T7 RNA-polymerase needs IPTG to be expressed) and therefore permits transcriptional modulation. Finally, the terminator chosen was L3S2P21 (BBa_K2675031).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 81
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



1. Usage and biology: reporter and fluorescence

This part is used inside BL21 competent cells, where we cotransform a plasmid containing the current part, and another containing the LbCas12a (BBa_K2927005) protein. To build our plasmids we used Golden Gate assemby, and to transform them into BL21 cells we used Heat Shock transformation. More details about the building process are available on our Biosensor library building page.

In order to determine if our sensors worked properly we had to first induce our plasmids expression using IPTG (100uM) and then lysate the cells for liberating our CRISPR-Cas construct. The method used for that was enzymatic lysis (using a buffer without EDTA). After lysing our cells we proceed with the detection.

As context we must point out that for our CRISPR-Cas ensemble to be a detection system, what we did was to add a reporter to the media where the Cas may become active. The basis relies on LbCas12a’s special property of collateral trans cleavage activity. That means that when it recognizes the specific target sequence determined by the designed gRNA with which it is coupled (thus becoming active) it unspecifically cuts not only the target DNA but also any dsDNA present in the media. In this way, whenever Cas is activated, fluorescence arises because the reporter is cut and releases the fluorescent molecule. All this is step-by-step indicated in Figure 1.

Figure 1: General Cas12a detection scheme. In numbers (1-4) are noted the fundamental steps for Cas12a assembly and dsDNA target detection. (1) Cas12a and gRNA assembly; (2) dsDNA target recognition and Cas12a activation; (3) Appearance of the collateral activity; (4) Signal detection due to reporter cleavage, which provokes liberation the fluorophore from the quencher giving rise to fluorescence.

Figure 1 Description: General schematic of Cas12-gRNA-reporter functioning containing the main enzymatic reactions. Cas12 binds to the gRNA, this complex gets activated by recognizing the specific target and cis-cleavage activity starts by cutting this dsDNA target. After that, cas12 trans-cleavage collateral activity cuts any nonspecific DNA fragment, cleaving the reporter. The reporter contains a quencher, which inhibits the fluorophore, and when cutted, luminescence comes out.


2. Characterization: model

CRISPR-Cas12 enzyme kinetics model was created by ARIA with the aim of understanding the dynamics and limitations of using the CRISPR-Cas technology for detection purposes. The model is extensively explained in our more complete composite part: BBa_K3791021, since the experimental parameters and results used for comparing with simulations were the ones performed with this part.

3. Characterization: Experimental results

3.1. Testing conditions

The fluorescence emitted by our system cannot be seen by naked eye, it needs to be checked with special light. Moreover, if we desire not only to determine its presence but also quantify it, we use special machinery as a Plate Reader. In our case, quantifying the amount of fluorescence resulting from our reactions was especially important as since we performed the detections in lysed bacterial cells there were lots of DNases, apart from Cas, cutting the reporter. The specifications for the configuration of the Plate Reader which are specified in Table 1 were designed by us, adapting the wavelength values to the ones specified in the reporter user manual. More details about measurement conditions are available in our Results measurement page.

Table 1. Plate reader configuration
Parameters Value
Temperature 37ºC / Room temperature
Duration 2h
Use kinetic interval Measure every 5 minutes
Excitation 530 nm
Emission 560 nm
Number of flashes 20
Measure from Bottom
Gain Optimal (100 RFA) + Use Gain Regulation


3.2. Best detection results

All experiments have been conducted under controlled conditions to respect procedural consistency and thus achieve the highest reproducibility of the results. For the results shown below, three biological replicates were used to ensure trustability.

The fluorescence results for this composite part + LbCas12a (BBa_K2927005) are shown in Figure 2.

Figure 2: Efficient gRNA Erythromycin testing results.
The results shown above as successful, a distinguishable difference between the positives and negatives can be seen. Peak value of and 32677 RFU is found in an optimal range. Therefore, the results confirm our engineered biological system serves as a biosensor and accomplish the purpose for which it was created.
[edit]
Categories
//collections/crispr-cas/diagnosis
//function/crispr/grna/construct
//function/crispr/grna/efficient
Parameters
None