Improvement of an Existing Part

Introduction

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The existing part that we used for our experiments and we decided to improve is the Cas13a Lbu (BBa_K2926001) designed by iGEM19_Bielefeld-CeBiTec team. This part codes for the Cas13a derived from Leptotrichia buccalis. According to the part's documentation in the registry 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.

A fundamental prerequisite for the practical application of recombinant enzyme-based diagnostic devices is the production of the enzyme in large quantities and in a functional form. However, protein expression is a complex process that involves hundreds of molecular components and is affected by many variables which need to be optimized for efficient protein production in large quantities (Liponska et al., 2018). The Cas13a protein derived from Leptotrichia Buccalis has a considerable molecular weight of approximately 138kDA and the effective recombinant Cas13a protein production in Escherichia coli can often be challenging.

Optimization approaches for protein production can be applied in different steps of the whole process, however, we decided to focus our efforts on optimization strategies related to the translation step of protein synthesis. Specifically, two approaches were followed for protein production optimization. The first strategy or else cis-optimization approach involves the usage of a DNA CDS sequence that is optimal for Cas13a production in E.coli based on codon metrics and codon frequency. The second approach or else trans-optimization involves the incorporation of the SUMO (small ubiquitin-related modifier) protein in fusion with Cas13a protein for efficient protein production in the soluble cytoplasmic function. The addition of the SUMO solubility tag in fusion with the Cas13a, could enhance protein expression and solubility, decrease protein degradation and simplify protein purification (Butt et al., 2005).

As described above, two different optimization approaches were followed for the improvement of the part BBa_K2926001:

1. Cis-optimization: codon optimization for efficient Cas13a production in ecoli.
2. Trans-optimization: generate gene chimera for Cas13a production in fusion with SUMO solubility tag.

Cis-optimization: codon optimization for efficient Cas13a production in E.coli.

In the context of synthetic biology, codon optimization is widely used to ameliorate gene expression in heterologous expression systems. Codon optimization is based on the basic principle of the genetic code that the distribution of the 64 unique DNA codons is non-random. Within a genome exist both rare and abundant DNA codons and their distribution varies across all organisms. The host-specific codon usage bias (CUB) influences translation efficiency especially when there is a high dominance of rare codons in the genetic sequence that is intended for translation. A common strategy for common optimization is the replacement of the rare codons with more frequently occurring ones, in accordance with the CUB of the specific organism.

Since we decided to express LbuCas13a protein in BL21 (DE3) E.coli strain, we followed a codon optimization approach to achieve maximum protein expression efficiency in the desired E.coli strain.

Trans-optimization: generate gene chimera for Cas13a production in fusion with SUMO solubility tag.

SUMO protein has been previously fused to the N-terminus of several proteins leading to increased expression and solubility of the protein of interest. But how does SUMO protein enhance protein solubility and proper folding? The answer lies in the structure of SUMO protein. Specifically, SUMO has an inner hydrophobic core and an external hydrophilic surface, thus exerting a detergent-like effect on proteins that cannot easily acquire their proper folding. Despite the advantages of SUMO addition, one drawback of this protein expression strategy is the necessary cleavage of the solubility tag. However, many SUMO proteases such as Ulp1, which are members of the cysteine protease superfamily can be used to cleave the SUMO tag without affecting the N-terminus of the desired protein (Panavas et al., 2009).

Therefore, by expressing the LbuCas13a protein in fusion with the SUMO-chaperone protein we aimed to enhance the quantity of Cas13a protein that accumulates to the soluble cytoplasmic fraction, succeeding proper protein folding and subsequent efficient SUMO tag removal.

Comparative experiments

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To demonstrate that our modifications had a positive effect on the efficiency of Cas13a protein production, we conducted comparative protein production experiments between the “improved” SUMO-LbuCas13a (BBa_K4170014) and the analogous protein from BBa_K2926001 part. For the implementation of the comparative experiments, we had to replace the "optimized" SUMO-LbuCas13a protein from the pSB1C3 plasmid with the Lbu Cas13a deposited from iGEM19_Bielefeld-CeBiTec team in the registry, keeping constant all the other regulatory elements of the genetic device. The detailed cloning strategy for the incorporation of the SUMO-LbuCas13a (BBa_K4170014) into the LbuCas13a coding device under T7 promoter (BBa_K4170016) is described on the corresponding part registry page and on the results page of our wiki.

Cloning strategy of Cas13a Lbu (BBa_K2926001) for comparative experiments.

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

Step 2. Golden Gate-based sevaBrick assembly

Golden Gate assembly of the PCR amplified LacI-promoter-RBS and Cas13a Lbu parts (BBa_K4170016) 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 final plasmid after the Golden Gate assembly contains the same DNA elements/features as the cloned final SUMO-LbuCas13a coding device under T7 promoter (BBa_K4170016) replacing the CDS of the codon-optimized SUMO-LbuCas13a (BBa_K4170014) with the CDS of Cas13a Lbu (BBa_K2926001). Furthermore, the CDS of the molecular chaperone SUMO sequence has also been removed. In addition, utilizing this cloning strategy we incorporated the 6xHis affinity tag at the N-terminus of the Cas13a Lbu (BBa_K2926001) to facilitate the efficient purification of the protein utilizing a Ni-NTA affinity purification methodology.

Expression of recombinant LbuCas13a proteins.

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. After overnight incubation, 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.

Analyzing the results from the SDS-PAGE gel electrophoresis and the Western Blotting (Figure 3) 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.

SDS-PAGE gel electrophoresis and Western blotting of "improved" SUMO-LbuCas13a protein (BBa_K4170014)

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.

Analyzing the results from the SDS-PAGE gel electrophoresis and the Western Blotting (Figure 4) we can conclude that the SUMO-LbuCas13a is detected in both soluble and insoluble fraction (Inclusion Bodies). The protein band which corresponds to the SUMO-LbuCas13a fusion protein is detected at the molecular weight of 155kDa. Therefore, we can assume that the addition of the SUMO solubility tag enhanced the soluble fraction of the LbuCas13a protein. The purification of soluble proteins is less expensive and time consuming compared to the process needed for protein purification and recovery from inclusion bodies. Utilizing the chaperone-mediated LbuCas13a protein recovery from soluble fraction ensures the integrity of the refolded proteins. The resolubilization procedures required to recover the protein from inclusion bodies can disturb the integrity of the protein. In addition, if we compare the Figures 4 and 5 which correspond to the Cas13a Lbu (BBa_K2926001) and SUMO-LbuCas13a (BBa_K4170014) respectively, we can understand that the codon optimization procedure enhanced the total protein yield in both the soluble and the insoluble fraction of the LbuCas13a protein. Further information about the downstream experiments regarding the SUMO-LbuCas13a purification with His SpinTrap purification columns and the enzymatic removal of the SUMO protein can be found on the results and on the experiments experiments pages.