Part:BBa_K4170056

Cas13a Lbu expression device

This expression device consists of the Cas13a Lbu protein under the transcriptional control of the T7 promoter. All regulatory elements essential for the efficient protein expression of the Cas13a Lbu protein are include in the device. The Cas13a Lbu protein is derived from Leptotrichia buccalis and the sequence was designed by iGEM19_Bielefeld-CeBiTec team. According to the part's documentation in the registry (BBa_K2926001) the EcoRI and PstI site have been removed to succeed RFC [10] compatibility. This part was used by the Bielefeld-CeBiTec team for assays in S.cerevisiae. iGEM22_Thessaloniki_Meta constructed this device as part of the protein expression comparative experiments with the SUMO-LbuCas13 codon optimized for E.coli.

Basic parts assembled for device construction

The part sample which is flanked at the beginning and the end with prefix and suffix respectively, is composed of the following basic parts assembled together in series and downstream of the prefix:

Bacterial terminator for LacI CDS (upstream of prefix): putative bacterial transcription terminator
Biobrick Prefix sequence (backbone sequence): BioBrick prefix for parts that do not start with "ATG"
LacI Coding sequence: Lac repressor
LacI promoter: Promoter that has the transcriptional control of Lac repressor
T7 promoter: promoter for bacteriophage T7 RNA polymerase
Lac operator: Lac repressor protein binding site
Ribosome binding site: efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
6XHis tags: 6xHis affinity tag
cas13a Lbu CDS (BBa_K2926001): Cas13a protein derived from Leptotrichia Buccalis
Biobrick Suffix sequence (backbone sequence) : universal suffix for all parts
His operon terminator (downstream of Suffix): putative transcription terminator from the E. coli

Figure 1. Cas13a Lbu expression device in pSB1C3 plasmid backbone

Cloning strategy of Cas13a Lbu (BBa_K2926001)

The CDS of Cas13a Lbu (BBa_K2926001) flanked by appropriate recognition sequences of BsaI restriction enzyme was ordered from IDT and cloned downstream of the T7 promoter in pSB1C3 plasmid with Golden Gate assembly. This part is the Cas13a Lbu part.

The cloning process is described in detail below:

Step 1. PCR amplification

PCR amplification with Ev and Pv standard primers from Basic SevaBrick Assembly (Damalas et al., 2020) using pSB1C3 (Bba_J36400-2022 DNA distribution Kit) a template (Figure 1). This PCR produces the pSB1C3 backbone part ready for Golden Gate assembly.

Figure 2. 1 % agarose gel electrophoresis of the PCR amplified pSB1C3 plasmid with Ev and Pv primers. The estimated DNA bands are 2029bp.

PCR amplification with cas13a T7 P0 FWD and SUMOLESS RVS primers using the LbuCas13a coding device under T7 promoter (BBa_K4170016-link) as a template (Figure 2). This PCR produces the LacI-promoter-RBS part ready for Golden Gate assembly.

Figure 3.1 % agarose gel electrophoresis of the PCR amplified LbuCas13a coding device under T7 promoter (BBa_K4170016-link) with T7 P0 FWD and SUMOLESS RVS primers.

Step 2. Golden Gate-based sevaBrick assembly

Golden Gate assembly of the PCR amplified LacI-promoter-RBS and Cas13a Lbu parts (BBa_K2926001) along with the ‘linearized’ pSB1C3 backbone part for the efficient construction of the LbuCas13a coding sequence under the transcriptional control of the Lac Repressor.

The Golden Gate assembly products underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using the primers VR and VF2. Picking sample from different colonies and then evaluating the results on a 1 % agarose gel electrophoresis we concluded that the Golden Gate assembly was successful in the first colony. The PCR product in the 1st colony has the suitable number of basepairs (5392bp), while the 2nd colony showed no PCR amplification. For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of dH20. The other half amount was picked for liquid overnight culture.

Figure 4.1% agarose gel electrophoresis of the PCR amplified product Cas13a Lbu-T7 (BBa K2926001) with VF2 and VR primers

Cas13a Lbu protein expression

Protocol for the expression of the recombinant Cas13a Lbu protein.

This protocol was followed for all the protein expression experiments with minor modifications at the IPTG concentration, induction temperature or time of induction .

The procedure of the protein expression initiates with the preparation of the bacterial pre-culture (15ml) incubated overnight at 37 °C. The following day the pre-culture was diluted in 1L of nutrient media, followed by a further incubation at 37°C of the diluted pre-culture until Optical Density (OD600) reached 0.7 – 0.8 (continuous measurements at the photometer at specific time points).
To induce LbuCas13a protein expression we added IPTG to the 1L bacterial culture to achieve a final concentration of 1mM. The bacterial culture was then incubated for 6 hours at 25°C and the bacterial culture was centrifuged at 10.000 g for 20 min at 4 °C and the supernatant was discarded. The bacterial pellets were resuspended in binding buffer (3oomM NaCl, 50mM NaH2PO4, 10mM imidazole, pH 8) followed by successive freeze-thaw cycles. After the freeze-thaw steps, Lysozyme (100mg/ml) and Triton x-100 were added and ultrasonication was performed to lyse the bacterial membrane. After ultrasonication, DNase (1mg/ml), MgCl2 (8mM) and Protease Inhibitor (PI) were added and the samples were incubated in a cold room for 2 hours on a rotator machine.
To obtain the soluble fraction of the protein, refrigerated centrifugation was carried out and the supernatant was then filtered by using 0.22 μm filter units. The remaining pellet constitutes the Inclusion Bodies (IBs), which also contains the insoluble form of the protein of interest. To isolate the SUMO-LbuCas13a protein from the IBs, the bacterial pellet is subjected to successive washing steps using 3 different buffers (A, B, C) followed by ultracentrifugation at 30.000g for 20 min at 4 C. The process is completed by resuspending the protein in L-Arginine solution which promotes the proper folding of the protein restoring its functional quaternary structure.

SDS-PAGE gel electrophoresis and Western blotting of Cas13a Lbu (BBa_K2926001)

Equal volumes of protein samples corresponding to the filtered soluble and resuspended insoluble (IBs) solution respectively, were loaded into each well of the SDS-PAGE gel for separation based on molecular weight. After gel electrophoresis completion, suitable images of the gels were obtained. After the separation of the protein mixture through the SDS-PAGE gel electrophoresis, it was transferred to the PVDF membrane for western blotting. After the initial blocking step, the addition of the anti-His primary and the anti-mouse alkaline Phosphatase-Labeled secondary antibody, the protein bands were detected using the alkaline phosphatase detection method.

Figure 5A. 8% SDS-PAGE electrophoresis of Cas13a Lbu (BBa_K2926001) protein after a 6h induction at 25°C using 1mM IPTG. Lane 1: protein marker (nippon genetics, bluestar prestained ), lane 2 : Cas13a soluble part, lane 3 : Cas13a insoluble part (IBs).B. Western blot of Cas13a after a 6h induction at 25°C using 1mM IPTG. Lane 1 : protein marker (nippon genetics blue star prestained), lane 2 : Cas13a soluble part, lane 3 : Cas13a insoluble part (IBs).

Analyzing the results from the SDS-PAGE gel electrophoresis and the Western Blotting (figure 5) we can conclude that the Cas13a Lbu (BBa_K2926001) protein is detected only in the insoluble fraction which corresponds to the inclusion bodies of the bacteria. The protein band which corresponds to the LbuCas13a protein is detected at the molecular weight of 130kDa. However, no LbuCas13a protein band is detected at the soluble cytoplasmic fraction. The process of obtaining bioactive functional protein from inclusion bodies is usually labor intensive and the yields of the recovered recombinant protein are often low.

Sequence and Features

Assembly Compatibility: