1.  Construction of the recombinant  plasmids

We prepared three DNA segments that can encode the sulfur-modified dependent restriction enzyme we want. They are Asp, Sva, and Sga, respectively. In itially, we needed to extract pET-28a(+) from our E. coli and test whether we got enough sample concentration. We used nanodrop and got an eligible concentration.

To express it in the competent DH5 αwe have to cut and insert it into the carriers. I nitially, enzyme digestion is done with pET-28a(+) to get our vector. The segments are inserted into the XhoI and NdeI  sites of the pET-28a(+) vector, respectively, by ligase T4. We did another enzyme digestion and ran gel electrophoresis to see whether we got the right recombinant plasmid ( Figure 1.1 ). Thankfully, we got the right bp for all three DNA segments. Sga ran to the position between 1000 bp and 1500 bp, which matches its calculated number of 1314 bp. Sva ran to the position between 1000 bp and 1500 bp, a bit forward than Sva, which matches its estimated number of 1230 bp. At the same time, Asp ran to the position around the 1000 bp line, which matches its calculated number of 939 bp.

 

F igure 1.1. Gel electrophoresis results of vectors and target gene fragments

After determining that we had constructed the correct recombinant plasmid, we retransformed it into BL21 receptor cells for subsequent protein expression. It is worth mentioning that the solid medium used was supplemented with an antibiotic (Kanamycin), which ensured that we screened to get the correct E. coli.

 

Figure 1.2. Growth of recombinant plasmids of Sga,Sva, and Asp after transformation of Escherichia coli in plate culture

For further confirmation, we sent samples of the bacterial fluid to the company for sequencing (Azenta). The sequencing results are shown below, which is 100% proof that we got the right plasmid. ( Figure 1.3 ).

 

F igure 1.3. Test sequence of Sga-pET28a, Sva-pET28a and Asp-pET28a

2. Protein expression and purification

After that, IPTG is added to induce protein expression. To get a pure target protein, we used Nickel column purification, and an SDS PAGE ( Figure 2.1 ) was done to show whether we have our target protein and whether it is purified or not.

 

Figure 2.1. SDS PAGE results of target protein after Nickel column purification

a: protein from solution with elution buffer

b : protein from solution with wash buffer

c: protein from supernate from ultrasonication

Figure 2.1 shows that  B and C are supposed to have little or no target protein, while A should have a single stripe. Sva shows no results in all three samples, meaning we failed to extract the protein of Sva. But we still got target proteins in samples A in Asp and Sga. Happily, the size of the protein obtained was in accordance with the expected values.

 

Figure 2.2. SDS PAGE results of target protein after concentration

Then, we use Bradford to test our samples' concentration so we can do quantitative delusion. We first get a standard   curve  with a formula by adding 1-6mg/ul BSA, a protein with known concentration as a contrast of concentration. The y-axis represents OD, and the x-axis represents protein concentration. We tested the Asp and Sga samples, respectively, by subtracting the background value from our data, which is the concentration of water, and putting the value of OD into the formula to get protein concentration. We prepare three samples of the same kind and take the average value from three repeating samples to get the most accurate concentration. The respective results are 0.853392145mg/ul for Sga and 0.164146188mg/ul for Asp. As the concentration for Asp was too low, we decided to continue our experiment with Sga.

 

Figure 2.3. S tandard   curve   of  BSA

   Since we failed to get the Sva sample and didn’t have enough concentration of Asp after nickel column purification, w e redid the protein purification of these two samples and ran SDS  PAGE.

 

Figure 2.4 SDS PAGE results of the target protein

(A) SDS PAGE of Asp ;(B) SDS PAGE of Sva

S: protein from supernate after ultrasonication

p: protein from precipitate after ultrasonication

Ni: Protein obtained after nickel column purification

Q: Protein obtained after ion-exchange chromatography

F rom Figure 2.4(A), we can see that there are all kinds of proteins in supernate after ultrasonication precipitate after ultrasonication because the results shown on them on SDS PAGE are scattered and with a large amount. However, after doing nickel column purification and ion exchange chromatography, concentrated lines on each sample indicate that the purification is successful, and we have only one kind of protein. We then checked the kDa of Asp, which is 36kDa, and the result is successfully confirmed as the line is just about the same level of scale 35.

We then repeated the same procedure in the Sva sample. F rom Figure 2.4(B), we can also see that there are all kinds of proteins in supernate after ultrasonication precipitate after ultrasonication because the results shown on them on SDS PAGE are scattered and with a large amount. However, after doing nickel column purification and ion exchange chromatography, concentrated lines on each sample indicate that the purification is successful, and we have only one kind of protein. We then checked the kDa of Sva, which is 45kDa, and the result is successfully confirmed as the line is just about the same level of scale 45.

3 Function Test

After extracting the target proteins, purification (nickel affinity chromatography, Q column chromatography, gravity column) and concentration were done, preparing for two function analyses: EMSA and nucleic acid cleavage test. T his part presents the overview and experiment results of the function test for the enzyme we obtained.

3.1 Electrophoretic mobility shift assays ( EMSA)

The EMSA test aims to test the binding specificity (phosphorothioate dependent in this case) of the Sga enzyme that is purified. E MSA 5x buffer is prepared with 100 mM Tris-Cl and 50 mM NaCl concentrations. A  10ul system is then used to achieve binding between the target enzyme and the dsDNA – phosphorothioate B7A and non-phosphorothioate BL21. The system consist s  of the following compositions. (Unit: ul)

Table 3.1 EMSA system (Unit: uL)

 

T he dsDNA is prepared from annealing of given ssDNA. Enzyme binding is followed by SDS PAGE (Sodium dodecyl sulfate – polyacrylamide gel electrophoresis). The product obtained is then sta ined using SYBR   Gold （ Invitrogen ）  without light, thus observed using a ge l imager. We expect enzyme binding with only ptDNA, thus no binding with non-ptDNA. The two result images are demonstrated in Figure 3.1 （ A ） (non-pt DNA) and Figure 3.1 （ B ） (mtDNA).

 

Figure 3.1 SDS-PAGE result

（ A ） R esult of BL21 EMSA （ B ） Result of B7A EMSA

A s shown in Figure 3.1, non-pt DNA BL21 presents no significant sign of successful binding with normal DNA chain length – around 25 bp. Non-pt DNA BL21 presents various DNA chain lengths under the SDS PAGE test, including a normal chain with a length of around 25 bp and chains with a length of over 250 bp. The significantly longer chains observed on every DNA protein level (not including the control level) represent successful binding between the target enzyme and phosphorothioate B7A DNA.

I n short, enzyme binding is observed in the ptDNA group while not observed in the non-ptDNA control group. T herefore, phosphorothioate-dependent binding of the target enzyme is confirmed.

Next shown are the results of our second round of experiments. The methodology used is the same as in the first round, and in this round of experiments, we obtained results for ASP and Sva.

 

Figure 3.2 R esult of BL21 and B7A EMSA for  SVA

(A) EMSA result of Asp ;(B) EMSA result of Sva

As shown in Figure 3.2, non-pt DNA BL21 presents no significant sign of successful binding with normal DNA chain length – around 25 bp. As with previous analyses, the EMSA results for Asp and Sva also demonstrated binding of phosphorothioate to the target enzyme

3.2 Nucleic acid cleavage test

The nucleic acid cleavage test aims to test the cleavage specificity (ptDNA dependent in this case) of the enzyme we obtained .

Cleavage 2x   buffer  is prepared with 40mM Bis-Tris, 100mM NaCl, 2mM DTT, and 2mM MnCl2 concentrations. Among them, Bis-Tris (pH6.0) and NaCl suitable pH and NaCl concentration for enzyme cleavage, MnCl2 provides the Mn2+ inducer while DTT maintains the oxidation state of Mn2+ cations. Next, a 10ul system is used to test  for cleavage of non-pt BL21 DNA and pt B7A DBA by enzyme Sga. The system composition is demonstrated in Table 3.2. (unit: ul)

Table 3.2 Cleavage system (unit: ul)

 

Enzyme cleavage is followed by enzyme digestion. Protein K is used to digest the enzyme Sga, avoiding potential influence in the following ag a rose  gel electrophoresis (AGE). The samples are run on AGE; the results are shown in Figure 3.3. We expect to see cleavage only on ptDNA while no successful cleavage on non-ptDNA

 

Figure 3.3 AGE r esult of BL21 & B 7A Nucleic Acid Cleavage

A s shown in Figure 3.3, the non-pt BL21 DNA group presents no significant sign of cleavage on 0 to 80 protein concentration levels with normal chain length > 15000 bp, while presents a relatively small amount of cleavage on 160 protein concentration levels with chain lengths around and less than 15000 bp. In contrast, the pt B7A DNA group presents normal chain lengths >15000 bp on 0 to 2.5 level while a relatively large amount of successful cleavage on 50 to 160 level (with significantly larger amount on 40 to 160 level).

I n short, on the 0 to 2.5 level, both groups showed no sign of cleavage; on the 5 to 20 level, only the ptDNA group showed successful cleavage, and on the 40 to 160 level, the ptDNA group showed a significantly larger amount of cleavage compared to non-ptDNA. T herefore, the phosphorothioate-dependent specific cleavage of the target enzyme is confirmed.

The following are the results of our second round of experiments. In this round of experiments, we successfully obtained the results of ASP and SVA.

 

Figure 3.4 Nucleic acid cleavage result s  of BL21 & B7A

(A)Result of Asp ;(B) Result of Sva

Like the previous analysis, the phosphorothioate-dependent specific cleavage of the target Asp and  Sva enzymes is confirmed.

4. Conclusion

  In conclusion, in the first round of experiments, we successfully completed the plasmid construction and functional testing of Sga. All our validations proved the reliability and accuracy of the results. Most importantly, the functionality and specificity of our product, sulfur modification-dependent restriction enzyme, were confirmed.

   Admittedly , we did not  complete the tests for Sva and Asp in the first round. This could be due to a number of reasons. For Sva, samples containing elution buffer did not show results in the SDS - PAGE after nickel column purification. This may be due to the protein being denatured and placed into a lower pH solution. However, in the case of Asp, we actually found the target protein in the concentrated SDS - PAGE but had to abandon testing it because the protein concentration was too low.

   Leveraging the learnings from our initial experiments, our second attempt proceeded far more seamlessly. Following the established protocol, we successfully obtained results for both Sva and Asp. However, it's important to recognize that our achievements thus far are incremental. While our current findings affirm the direction of our research, certain aspects of our results warrant refinement. The need for repeated trials remains paramount to bolster data accuracy.

   Moving forward, once we are confident in the precision of our existing experimental data, we intend to supplement our research with further functional data related to our primary product: the sulfur-modification-dependent restriction enzyme.