Part:BBa_K5507008
sacB-5-Ptac-pgi-rrnB-T-sacB-3
For overexpressing the pgi protein in K. xylinus. The Ptac-pgi-rrnB-T construct serves as the expression frame for the pgi gene, while sacB-5 and sacB-3 are the flanking sequences of the sacB gene.
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 785
Illegal BglII site found at 3037 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3739
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2781
a. Construction of BBa_K5507008
For the overexpression of the pgi gene from E. coli, the sacB locus, which encodes levansucrase, was selected as a homologous recombination site because knocking out this gene has no metabolic effects under glucose conditions. First, the kana expression cassette will replace the sacB gene locus as a selectable marker. Then, pgi will replace the kana gene, resulting in the loss of kana resistance, confirming successful gene insertion.
Before constructing BBa_K5507008, we obtained the sacB-5-Ptac-kana-rrnB-T-sacB-3 (BBa_K5507009) component using restriction enzyme digestion and T4 DNA ligase methods (Figure 2). Based on this component, we amplified the rrnB-T-sacB-3-pGEM-T-sacB-5-Ptac fragment through PCR and the pgi gene from the K. xylinus genome. Next, we employed Gibson assembly (ClonExpress II One Step Cloning Kit, Vazyme, China) to connect the two fragments via homologous arms (Figure 3), resulting in BBa_K5507008.
b. Construction of K. xylinus pgi OE
Firstly, the plasmids pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 (BBa_K5507009) was introduced into K. xylinus, located at the sacB gene locus via electroporation. The resulting sacB knockout mutant (ΔsacB) was screened for kanamycin resistance and confirmed by PCR (Figure 4).
ΔsacB will gain resistance to kanamycin. Subsequently, introducing BBa_K5507008 into the bacteria will lead to the loss of kanamycin resistance, resulting in the pgi overexpression (OE) strain. When constructing the pgi-OE gdh-KO strain, transforming BBa_K5507010 into the pgi OE strain will restore the kanamycin resistance gene. The resulting strain was screened for non-kanamycin/kanamycin resistance and confirmed by PCR (Figure 5).
c. Growth curves of K. xylinus pgi OE
First of all, we wanted to make sure that our engineered K. xylinus can still grow normally. WT and pgi OE strains was inoculated into flasks containing liquid HS media with 0.1% cellulase and cultured at 30 ℃, 180 rpm. On 0, 1, 2, 3, 4 and 5d, the OD600 were measured using a NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA, USA).
Two strains had similar growth rates, which meant that the overexpression of pgi did not dramatically influence cell metabolites. Strains pgi OE showed a slowly growth than others before reached the stationary stage. This might be due to the matter and energy used to reproduce being moved to the overproduction of bacteria cellulose (figure 6).
d. Bacterial cellulose (BC) production assay
Then, we proceeded to test bacterial cellulose production. The wild-type K. xylinus and the engineered strains were inoculated into HS media with 1% ethanol and cultured at 30°C with shaking at 180 rpm. Bacterial cellulose production occurs alongside bacterial growth and proliferation, ultimately forming a mass enveloped by BC. After 7 days, following a simple purification process, the BC yield in the media was measured.
The results confirmed that the WT can produce a relativity small amount of BC. The overexpression of pgi gene increased BC production by about 111%. Compared to single mutant, pgi-OE gdh-KO showed massive BC overproduction, increased contents by 227% (Figure 7).
e. Water retention performance assay of BC
BC possesses excellent physicochemical and mechanical properties, such as high purity, high crystallinity, strong water retention capacity, high degree of polymerization, large surface area, and good chemical stability. Additionally, compared to other water retention materials like hydrogels, polyacrylamide (PAM), and sodium carboxymethyl cellulose (CMC), BC offers biocompatibility, biodegradability, and renewability. As a more cost-effective and sustainable solution, BC is particularly suitable for regions experiencing rapid desertification and severe soil moisture loss. Its three-dimensional structure and adaptability to various environmental conditions make it an ideal material for improving soil water retention.
However, there is currently no data available on the water retention effects of BC in soil. To address this, we transplanted six seabuckthorn (Hippophae rhamnoides) seedlings, each with a similar initial growth state and a height of about 1 meter. Seabuckthorn is a species known for its wind resistance and economic value. Three of the seedlings were transplanted without adding BC, serving as the control group, while the other three were treated with BC, forming the BC group. Starting with similar soil moisture levels, we withheld watering and continuously measured the soil moisture content, resulting in the following data (Figure 8).
At the start, the soil moisture content for all seedlings was around 68%. Over time, as water evaporated and was absorbed by the seedlings' roots, a clear difference in moisture retention between the groups became apparent. The BC group's water loss rate was noticeably slower than that of the control group. After 7 days, the BC group still maintained about 33% moisture content, while the control group's moisture content had dropped to 16.5%, which is below the minimum required for the seedlings to survive (Figure 8). Therefore, the application of BC during tree planting helps to slow soil moisture loss.
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