Team:NNU-China/WetLab/Results



Results



1. Construction of the BL21 (DE3)-derived Variant Strains Library Using Base editor

What work we have done:

· A tailored RBS sequence of 5'-GGGGGGGG-3' was integrated into the corresponding site of the T7 RNAP, yielding a starting strain of BL21 (DE3)-RBS8G. (please see the part of engineering success)
· The dual plasmids system of CBE can successfully work in the BL21 (DE3). (please see the part of engineering success)

Results:

    Based on the dual plasmids system of CBE, the library of BL21 (DE3)-derived variant strains were constructed. The pCBE and pTargetS plasmid were transformed into the starting strain of BL21 (DE3)-RBS8G. 90 single colonies were randomly selected for sequencing, which found 48 variants with different RBS sequences with an editing efficiency of 53%. However, it is noteworthy that the transformants were grown on the solid medium for a longer time, the reproducibility of the results gradually increased, and the majority of variant RBS sequences became GAAAAAAG (Fig. 1), probably due to the continuous base editing in the transformants. The above results show that although CBE possesses the advantages of simplicity and rapidity, editing results and efficiency applied in BL21 (DE3) are not sufficiently stable.
Fig. 1. Schematic representation of the changes in G and A abundance of the RBS variant sequences of T7 RNAP obtained from CBEs experiments.

2. Construction of the BL21 (DE3)-derived Variant Strains Library Using Base editor

What work we have done:

· The pTarget+Donor plasmid was successfully constructed, and then the Donor DNA plasmids library was successfully built by megawhop PCR. (please see the part of engineering success)
· The lacI gene in DE3 region was replaced with the antibiotic gene of chloramphenicol (CM), yielding the starting strain of BL21 (DE3)-CM. (please see the part of engineering success)

Results:

    The sgRNA on the pTarget+Donor was designed to target the gene of CM. the pCas and pTarget+Donor plasmids were co-transformed into BL21 (DE3)-CM via electroporation, then those of transformants that cannot grow under CM condition were selected as positive transformants for sequencing. Results showed that a total of 284 single colonies were grown on 10 plates, of which 90 were randomly selected for sequencing analysis. Surprisingly, 71 different variants were identified from 90 simples, which indicated the efficiency of obtaining variants is up to 78.9%. It was hypothesized that roughly 224 different variants could be obtained from approximately 284 single colonies, reaching 87.5% of the theoretical values (28=256). Compared to the CBE method, it is clear that the CRISPR-Cas9 system is more efficiently edited and more suitable for the construction of the BL21 (DE3)-derived variant strains library, despite both the same transformation method and screening time. Moreover, in order to exclude the impact of batches, we subsequently perform three batches, respectively. 25 of variant clones after sequencing were selected from each batch for analysis (Fig. 2), and different variants were found in different batches, which proved the stability of this method. By comparing the RBS sequences in the different variants, it was found that the probability of G and A bases was related to the position.
Fig. 2. Partial sequences and the G and A abundance variation maps of RBS variants obtained by CRISPR from three batches respectively.

3. Assessment of the Quality of the BL21 (DE3)-derived Variant Strains Library

What work we have done:

· The pET-GFP plasmid was successfully constructed. (please see the part of engineering success)

Results:

    The change of translation level of T7RNAP was measured using eGFP fluorescence intensity per OD600. We constructed the plasmid of pET-eGFP, and transformed into the library of BL21 (DE3)-derived variant strains. Compared to the wild strain, the fluorescence intensity in the library ranged from 28% to 220%, demonstrating that RBS sequences change of T7 RNAP significantly affect the expression level of the target gene on the pET plasmid. And, single colonies growing on Kan resistant plates were randomly selected and cultivated in 96-well plates for testing the fluorescence intensity (Fig. 3A). Furthermore, 45 representative strains, depending on the fluorescence intensity, were transferred into tubes for more rigorous validation (Fig. 3B). It was found that the value of tube fermentation matched that of 96-well plate, implying that the 96-well plate could be used to carry out a preliminary screen for high expression of the target gene.
Fig. 3. A. the fluorescence intensity of randomized different strains in the 96-well plate. B. the fluorescence intensity of 45 of BL21 (DE3)-derived variant strains in shake flask.

4. Development of High Throughput Host Screening Platform and its Application in AMPs production

What work we have done:


· The plasmid of pET-AMP- eGFP was successfully constructed and tested.

Results:

    Based on the library of BL21 (DE3)-derived variant strains, the high throughput host screening platform for AMPs production was constructed. In this system, the target AMP was fused with the eGFP and was transferred into the library of BL21 (DE3)-derived variant strains, then the best host was screened by fluorescence intensity in 96-well plates. Using this platform, it takes only three days to obtain the highest-expression host. In the part engineering success, the production SMAP has been greatly improved. Here, we tested another 9 kinds of AMPs, as shown in Table 1.
Table 1. 9 kinds of AMPs.
Name Type Description
BBa_K3868100 Coding MME
BBa_K3868101 Coding Fa-AMP1
BBa_K3868102 Coding Alloferon-1
BBa_K3868103 Coding Oxysterlin 1
BBa_K3868104 Coding LL-37
BBa_K3868106 Coding DCD-1L
BBa_K3868107 Coding Coding SPINK9
BBa_K3868108 Coding PEW300
BBa_K3868109 Coding ID13

    Through screening 96-well plates of ten antimicrobial peptides, we selected three of the best production hosts from each AMP production and transferred them to cone bottles containing 30 mL TB medium for 20 hours of fermentation culture for expansion verification. Results showed that all of AMPs were significantly increased. In particular, the average production for MME, DCD, ID-13 has increased by 27 times, 50 times, and 40 times, respectively.
Fig. 4. A. The unit cell fluorescence intensity of MME (BBa_K3868100) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture MME.

Fig. 5. A. the unit cell fluorescence intensity of Fa-AMP1 (BBa_K3868101) in the 96-well plate. B. the unit cell fluorescence intensity of the fermented culture Fa-AMP1.

Fig. 6. A. The unit cell fluorescence intensity of Alloferon-1 (BBa_K3868102) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture Alloferon-1.

Fig. 7. A. The unit cell fluorescence intensity of Oxysterlin 1 (BBa_K3868103) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture Oxysterlin 1.

Fig. 8. A. The unit cell fluorescence intensity of LL-37(BBa_K3868104) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture LL-37.

Fig. 9. A. The unit cell fluorescence intensity of DCD-1L (BBa_K3868106) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture DCD-1L.

Fig. 10. A. The unit cell fluorescence intensity of SPINK9 (BBa_K3868107) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture SPINK9.

Fig. 11. A. The unit cell fluorescence intensity of PEW300 (BBa_K3868108) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture PEW300.

Fig. 12. A. The unit cell fluorescence intensity of ID13 (BBa_K3868109) in the 96-well plate. B. The unit cell fluorescence intensity of the fermented culture ID13.

5. Purification of fusion protein and identification of antimicrobial activity

Results:

    Furthermore, the AMP of MME (BBa_K3868100) and DCD-1L (BBa_K3868106) were chosen to purify and identify their antimicrobial activity. The fusion protein resulted from bacterium lysis was purified by Ni2+ affinity chromatography column, and then tested with 5% spacer gel and 15% separating gel for SDS-PAGE. A clear strip of about 27 kDa was obtained, which is consistent with the expected protein size. Freeze-drying is widely used in the pharmaceutical industry for the manufacture of protein drugs. About 30.4 and 42.3 mg of MME and DCD fusion protein powder were obtained per liter of culture medium, respectively. Furthermore, the thrombin was used to isolate the AMP and eGFP protein. Purified products were experimented for antimicrobial activity with agarose hole diffusion method. It was indicated from the results that purified heterozygous peptides showed significant antimicrobial activity on E. coli.
Fig. 13. A.SDS‐PAGE results of MME-37-eGFP and DCD-14-eGFP protein in BL21 (DE3).B. MME and DCD was proved by the means of inhibiory zone to be able to kill the E. coli. (1:H2O; 2:Kan(2.5g/L); 3:AMP(4g/L); 4:MME(3.04g/L); 5: DCD(4.23g/L)).






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