Part:BBa_K4099006

pNCas9-LSEI-2094

Usage and Biology

Restriction endonuclease is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up foreign DNA in a process called restriction digestion; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifies the prokaryotic DNA and blocks cleavage. Together, these two processes form the restriction modification system. The plasmid, pNCas9-LSEI-2094 equipped with a CRISPR-Cas9 complex in order to kick out the DNA segment, LSEI-2094 gene. This gene is involved in the synthesis of an enzyme that is essential in the restriction-modification system. After this modification, the restriction enzyme could be temporarily inactivated so that the transferred exogenous DNA could successfully avoid the restriction effect of the host bacteria restriction system.

Figure1. Principle diagram of CRISPR-Cas9..

Construct design

Figure 2 shows the design of a CRISPR-Cas9-based gene knockout vector in L. casei ATCC 334. Single plasmid CRISPR-Cas9 system is applied, namely gRNA, Cas9 effector protein, and repair template on one plasmid.

Figure 2. CRISPR-Cas9-based gene knockout vector design in Lactobacillus casei ATCC 334. P is the promoter (promotor); T is the terminator (terminator); pNCas9 is the Cas effector protein; HA-L and HA-R are the left homology arm and the right homology arm, respectively..

sgRNA+HR+pNCas9 backbone is a key functional factor that kicks out the DNA segment, LSEI-2094 gene. The sgRNA and HR are inserted in the pLCNICK vector. (Figure 3 and 4).

Figure 3. Schematic map of pNCas9-LSEI-2094 expression plasmids..

Figure 4. A: DNA profile of the plasmid pLCNICK; B: DNA Profile of LSEI-2094+ upstream and downstream homologous arms..

Usage and Biology

BBa_K4099000 is a piece of RNAs that function as guides for RNA- or DNA-targeting enzymes, which they form complexes with.

Usage and Biology

BBa_K4099001 is a coding sequence of Cas9, an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence

BBa_K4099002

Usage and Biology

This is a coding sequence of left homology arm, refers to the flanking sequence on one side of the LSEI-2094 sequence to be inserted on the target vector and is used to identify and recombine the region.

BBa_K4099003

Usage and Biology

This is a coding sequence of right homology arm, refers to the flanking sequence on one side of the LSEI-2094 sequence to be inserted on the target vector is used to identify and recombine the region.

BBa_K4099005

Usage and Biology

This is a plasmid backbone of pLCNICK, which is the genome editing for Lactobacillus casei Lc2W.

Experimental approach

1.Electrophoresis result after PCR

Figure 5. Electrophoretogram of pLCNICK enzyme digestion and PCR result..

1-4 are pLCNICK after enzyme digestion, 5 is pLCNICK plasmid before enzyme digestion, 6~7 are upstream homologous arms after PCR, 8~9 are downstream homologous arms after PCR. This step is used to get the plasmids pLCNICK digested by enzyme XbaI and ApaI and gene HA-L and HA-R by PCR method for later in the process. Clean-up the product of pLCNICK, HA-L and HA-R to obtain pLCNICK backbone and HA-L and HA-R-fragments. Besides, we can get sgRNA-fragment by PCR method. pLCNICK backbone, HA-L and HA-R-fragments and sgRNA-fragment were connected by homologous recombination method.

2.Verification result of colony PCR

Figure 6. PCR verification result..

As showing above, there are bands (1~3, 6, 8~12) at 1000 bp around which are consistent with the DNA profile of the downstream homologous arms. Therefore, it indicated that we have successfully transformed the plasmid in E. coli.

Proof of function

After we electrotransformed the plasmid pIB165 as the foreign DNA to test the transformation efficiency of our modified L. casei, it took several days to culture and finally saw the comparison results. As showing above, we can see that the modified L. casei (KO) has higher transformation efficiency with remarkably more colonies than the wild (Wild).

Figure 7. Comparison between the wild L. casei (left) and modified L. casei (right, LSEI-2094 knocked out) in transformation..

Graph 1. Comparison between the wild L. casei and modified L. casei in transformation

Figure 8. Histogram comparison between wild strain and KO strain..

In addition, we measured OD600 of these strain groups which were pre-spread plates with different volumes of bacteria solutions so as to quantify the transformation results as showing above (Fig. 8). In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as the recombinant carrier in various areas. In order to scientifically determine the transformation efficiency of our modified L. casei (KO) and the wild L. casei (Wild), we collected the colony cultured which were pre-spread plates with different volumes of bacteria solutions and measured their OD600 after cultured for the same hours. In the meantime, we also aimed to explore the optimal condition for our modified L. casei’ growth by applying different amounts of the bacteria seed solution.

Table 1. OD600 of cultured L. casei..

According to the scatter plots, we chose to use the quadratic polynomial equation to build the model. After calculation, below are the constants of the solved quadratic polynomial equations of the KO group and Wild group, respectively.

Table 2. Model results..

Figure 9. Comparison between two fitting curves of KO group(red) and Wild group(blue)..

In Figure 9, we can clearly see that the modified L. casei shows much higher transformation efficiency than the wild L. casei especially when the volume of the initial bacteria seed solution is used less than 100 uL when the difference between them is remarkable.

Besides, the equation model we built for the modified L. casei as showing below, could be used to analyze the relationship between the volume of the bacteria seed solution and its OD600, it could be used as a reference when we conduct the expression efficiency tests in the future. If we could further build the relationship between the expression level and OD600, we could adjust the volume of the bacteria seed solution for culturing accordingly.

Improvement of an existing part

Compared to the old part BBa_K2201030, which is type II CRISPR RNA-guided endonuclease Cas9 from Streptococcus pyogenes, we designed a new part BBa_K4099006. We optimized the codon of NCas9 protein according to the preference of L. casei. Group iGEM17_Bielefeld-CeBiTec designed the CFP-YFP test system and changed the coding sequences of both of the fluorescent proteins using CRISPR-Cas9 method. We constructed a new part BBa_K4099006 equipped with an optimized NCas9 protein that cuts and removes a selected segment of the L. casei DNA. Our modified L. casei has much higher efficiency of the foreign plasmid transformation than the wild.

Table 2. Model results..

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 13411
  Illegal XbaI site found at 14374
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 8143
  Illegal NheI site found at 13795
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 6903
  Illegal BglII site found at 7540
  Illegal BglII site found at 13141
  Illegal BglII site found at 13195
  Illegal BglII site found at 14371
  Illegal XhoI site found at 3061
  Illegal XhoI site found at 7536
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 13411
  Illegal XbaI site found at 14374
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 13411
  Illegal XbaI site found at 14374
  Illegal AgeI site found at 6696
  Illegal AgeI site found at 14076
  
• 1000
  COMPATIBLE WITH RFC[1000]