Difference between revisions of "Part:BBa K5072007"

Latest revision as of 04:22, 30 September 2024

pEcgRNA-P57-2

Sequence and Features

Composite part BBa_K5072007 (pEcgRNA57-2)

Construction Design

pEcgRNA57-2 was amplified with the BBa_K5072005 (pEcgRNA29-2) plasmid using primers and replacing the N20 part of the primer, and was digested with DNA endonuclease DpnI at 37℃. The digestion product was then transformed into competent E.coli DH5α and cultured on a plate containing spectinomycin resistance smr. The plasmid profile of pEcgRNA57-2 is shown in Fig 1.

Fig 1. The plasmid profile of pEcgRNA57-2

Engineering Principle

We added the designed 20-base sequence to the primer and used the laboratory-stored BBa_K5072005 (pEcgRNA29-2) plasmid for reverse PCR to obtain the complete plasmid we want by replacing the N20 part of the original plasmid.

Experimental Approach

The length of pEcgRNA57-2 after amplification was 2248 bp. From the results of agarose gel electrophoresis, the amplified fragment met the expected length. We then used seamless ligase to connect and obtain the complete pEcgRNA57-2 plasmid.

Fig 2. Agarose gel electrophoresis of pEcgRNA57-2

We transformed the successfully constructed pEcgRNA57-2 plasmid into E.coil DH5α and obtained the corresponding monoclonal plate after overnight growth (Fig.3B). Then we selected three monoclonal colonies and performed colony PCR verification. Under the amplification of the corresponding primers, the length of the resulting band was consistent with the expected size, indicating that our fragment connection was successful (Fig.3C). Finally, in order to ensure that there was no base mutation or deletion in the plasmid, we sent the plasmid to a biological company for sequencing, and the results showed that the site of the connected fragment was successful (Fig.3D).

Fig 3. A: pEcgRNA57-2 plasmid spectrum, B: pEcgRNA57-2 plasmid transformed into DH5α monoclonal plate, C: monoclonal plate colony PCR results, D: pEcgRNA57-2 key fragment sequencing results comparison chart

After confirming that the plasmids were not problematic and could be replicated in large quantities, we also transformed the pEcgRNA57-2 plasmid into E. coli MG1655.

Fig 4. E. coli MG1655 transformed with pEcgRNA57-2

Characterization/Measurement

Two Layer Plating Method

Judging from the results of the double-layer plate experiment, compared with the control, all three treatments significantly improved the resistance of MG1655 to T7 phage (Fig.5). Furthermore, the resistance of pEcgRNA57-1 to T7 phage has reached two orders of magnitude, while the effect of pEcgRNA57-2 cutting the second site and simultaneously cutting the two sites of the P57 gene has even reached 100%. However, since the plaque count of the double-layer plate experiment depends on direct observation and there is a certain error, whether complete resistance has been achieved still needs to be further verified at the genetic level using molecular biological methods (P < 0.001) (Fig.6).

Fig 5. Double-layer plate experiment for cutting T7 bacteriophage P57 gene

Fig 6. The ability of the tested bacteria to resist infection by T7 phage under different treatments, (P < 0.001): Indicates that the result is extremely significant.

Growth Curve Assay

We observed two single-site cuts and one double-site cut of the T7 phage P57 gene, as well as the changes in OD600 within 0-16 hours after adding T7 phage. We set different MOI values, 0.02, 0.2, and 2, and the absorbance of the test bacteria under different treatments will also be different. The specific values are shown in Table 1-3. Then we use these data to draw a line graph.

Table 1 OD600 values of the tested bacteria under different treatment conditions of T7 phage P57 gene extraction at moi of 0.02

Table 2 OD600 values of the tested bacteria under different treatment conditions of T7 phage P57 gene extraction at moi of 0.2

Table 3 OD600 values of the tested bacteria under different treatment conditions of T7 phage P57 gene extraction at moi of 2

From the line graph, we can see that Escherichia coli MG1655 with the T7 phage P57 gene cut out, whether it is single-site cutting or double-site cutting, also played a certain resistance role, and in the double-layer plate, the effects of cutting the P57 gene at site 2 and cutting at both sites were almost the same, and the performance in the growth curve also showed the same trend. Therefore, compared with cutting the P29 gene, cutting the P57 gene may be a better choice to improve Escherichia coli MG1655's resistance to T7 phage infection. However, it is surprising that the modified bacteria with the first site cut are not as good as the negative control (T7 phage infection is added directly without modification) in the bacterial growth curve. This situation is most likely caused by phage contamination or a volume error during the addition process (Fig.7).

Fig 7A: OD600 values of the test bacteria under the treatment conditions of different excisions of T7 phage P57 gene when moi was 0.02, B: OD600 values of the test bacteria under the treatment conditions of different excisions of T7 phage P57 gene when moi was 0.2, C: OD600 values of the test bacteria under the treatment conditions of different excisions of T7 phage P57 gene when moi was 2

References

[1] Sapranauskas, R. et al. (2011) The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282

[2] Briner, A.E. and Barrangou, R. (2014) Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity. Appl. Environ. Microbiol. 80, 994–1001