Part:BBa_K5190003
pGEX-4T-1-PK
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 956
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 930
Illegal XhoI site found at 951 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2089
Illegal SapI.rc site found at 342
Illegal SapI.rc site found at 3171
Existing Part: PK (BBa_K3832010)
Summary
Compared with the old part of pyruvate kinase (PK, BBa_K3832010), we have increased the application range of pyruvate kinase, and probiotics can produce pyruvate kinase to produce short-chain fatty acids to help treat depression. And we added a new part pGEX-4T-1-PK (BBa_K5190003) and a lot of test data. The experimental test data are mainly the following aspects：
1.We constructed the recombinant plasmids pGEX-4T-1-PK, used heat shock transformation into E. coli DH5α to preserve the plasmids.
2.We performed protein expression in E.coli BL21. SDS-PAGE was used to identify whether the target protein was successfully expressed. Building upon the successful expression of the target protein.
3.We conducted in vitro enzyme activity assays for pyruvate kinase (PK) to more intuitively demonstrate the changes in enzyme activity of our engineered strains. Finally, we transformed the recombinant plasmids (PGEX-4T-1-PK) into E. coli ECN and used high performance liquid chromatography (HPLC) to detect the content of propionate.
Engineering Principle
Propionic acid salt levels in the intestine are believed to be associated with various diseases, including depression. Attempts to regulate gut microbiota to increase propionic acid salt levels to improve conditions are not uncommon. Once successful, such research will benefit hundreds of millions of people globally. Short-chain fatty acids (SCFAs) refer to fatty acids containing 5 carbon atoms or fewer, such as formic acid, acetic acid, propionic acid, butyric acid, and valeric acid, which are typically present in the intestine in salt form1. Current research focuses mainly on acetate, propionate, and butyrate salts. Due to the functional overlap and interconversion among these salts, supplementation of a single component can also effectively supplement short-chain fatty acid salts2.
We have analyzed the metabolic pathways and identified the key pyruvate kinase (PK) related to SCFAs (Figure 1). We aim to enhance the production of short-chain fatty acids in the strain by overexpressing the key pyruvate kinase (PK) in order to further achieve the purpose of auxiliary cure of depression.
Construction Design
The pGEX plasmid was provided by our institution's strain repository, and the target gene PK(BBa_K3832010) was synthesized by a biotech company. Using the pGEX plasmid as a template, the PK fragment was homologously recombined with a linearized plasmid to construct pGEX-4T-1-PK (Figure 2).
Experimental Approach
We constructed pGEX-4T-1-PK using homologous recombination. We amplified the pGEX-4T-1 vector backbone using PCR, with a length of 4969 bp (Figure 3A). We also amplified the PK target gene by PCR, with a length of 1455 bp (Figure 3B), indicating that the target gene and vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained using homologous recombination.
The recombinant plasmid was introduced into E. coli DH5α. We selected 10 colonies for colony PCR verification. Figure 4A shows a band consistent with the target fragment. Figure 4B shows a single colony on the plate. According to the results shown in Figure 4C, the gene PK was successfully connected to the pGEX-4T-1 vector without any obvious mutations, confirming the successful construction of the recombinant plasmid pGEX-4T-1-PK.
(A: Colony PCR. B: Clones on plate. C: Sequencing comparison)
After successful verification, we transformed the recombinant plasmid into E. coli ECN and spread the colonies on a plate. We selected six colonies for PCR verification. Figure 5A shows the colony PCR results, and Figure 5B shows the clones on the plate.
(A: Colony PCR. B: Clones on plate)
Characterization/Measurement
1. Protein Expression
We transformed the correctly sequenced plasmid pGEX-4T-1-PK into E. coli BL21 to verify protein expression. The size of the target protein PK is 54.1 kDa. Figure 6A shows protein bands of PK in both crude and purified protein, confirming successful expression. Similarly, we observed the same results in E. coli ECN (Figure 6B).
(A: E. coli BL21. B: E. coli ECN)
2. Functional Test
Ⅰ: Determination of PK Activity
The determination of PK enzyme activity was performed using the PK Activity Assay Kit from Solarbio. The main principle is that PK catalyzes the production of ATP and pyruvic acid from phosphoenolpyruvate and ADP. Lactate dehydrogenase further catalyzes the generation of lactate and NAD+ from NADH and pyruvic acid. The activity of PK can be measured by the rate of NADH decline at 340 nm.
Initially, we measured the PK activity of the control strains and found that they exhibited decent enzyme activity even without modification (Figure 7). After our modification, the PK enzyme activity of the strains increased by 1.1-fold at 4 °C and by 1.6-fold at 37 °C. These results indicate a significant enhancement in PK enzyme activity. Through experimentation, we confirmed that our modification resulted in a notable improvement in PK enzyme activity in the strains.
Ⅱ: Determination of Propionate Content by HPLC
In order to visually monitor the changes in acetate content of the engineered strains, we conducted fermentation tests on both the original and modified strains to detect the actual variations in acetate levels. Initially, the modified strains were activated twice and fermented in 100 mL conical flasks with an initial OD600 of 0.2 in a 30 mL fermentation system. When the OD600 of the strains reached ~0.8, we added inducer IPTG at a final concentration of 50 mg/L. Subsequently, we intermittently sampled the fermentation broth to measure the propionate content. Propionate was determined using an HPLC (UltiMate 3000 HPLC, Thermo Scientific) with a Bio-Rad Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) at 65 ℃ in an index detector.
We set different time intervals to determine the propionate content at 37°C. The HPLC experimental results are shown in Table 1. The data were plotted using GraphPad Prism software.
Table 1. Propionate content at 37°C
| Time | Propionate in the experimental group (mg/L) | Propionate in the control group (mg/L) |
|---|---|---|
| 0h | 0.00 | 0.00 |
| 8h | 612.20 | 570.77 |
| 24h | 694.62 | 594.01 |
| 36h | 706.65 | 631.88 |
| 48h | 817.57 | 670.77 |
As shown in Figure 8, the propionate content in both the control group and the experimental group at 37°C increased over time. The greatest change in propionate content occurred during the 0-8 hour period. The experimental group showed a significantly higher propionate content than the control group. According to the literature, E. coli itself can produce a small amount of propionate, which is consistent with our results3. After PK expression, the propionate content was higher than that of the control group, indicating that PK enhances propionate production.
Learn
1. This plasmid theoretically only increases acetate production, but we have not studied the impact of increased acetate production on other metabolic pathways. This aspect may have relevance for our future modifications and investigations. Understanding how the enhanced acetate production affects other metabolic pathways could provide valuable insights for our future research and modifications.
2. In our fermentation process, we added inducers. We can explore more environmentally friendly production methods by replacing the inducers with external regulatory conditions, such as temperature and light exposure, to induce expression through these greener and harmless approaches.
Reference
1.Jitrapakdee, S. & Wallace, J.C. Structure, function and regulation of pyruvate carboxylase. Biochem J 340 ( Pt 1), 1-16 (1999).
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