Difference between revisions of "Part:BBa K5507010"

Latest revision as of 02:59, 30 September 2024

gdh-5-Ptac-kana-rrnB-T-gdh-3

For the knockout of the gdh protein in K. xylinus. The Ptac-kana-rrnB-T construct serves as a marker gene in the genome to indicate whether gene recombination has been successful. gdh-5 and gdh-3 are the flanking sequences of the gdh gene.

Sequence and Features

a. Construction of BBa_K5507010

To construct a gdh gene knock-out strain, we constructed an expression cassette containing the Kana gene flanked by homology arms, each about 700 bp in length, on either side of the gdh gene. The upstream and downstream homology arms of the gdh gene were amplified by PCR from K. xylinus genomic DNA. The upstream flanking region was amplified using the primers gdh-gibson-gdh5-F and gdh-gibson-gdh5-R, while the downstream flanking region was amplified using the primers gdh-gibson-gdh3-F and gdh-gibson-gdh3-R. For expression, the fragment containing tac promoter, kana and rrnB-T terminator were amplified from BBa_K5507009 using the primers gdh-gibson-pkr-F and gdh-gibson-pkr-R. The plasmid backbone was amplified from the same part using the primer gdh-gibson-vector-F and gdh-gibson-vector-R (Figure 1, 2A).

These four fragments were ligated using the Gibson assembly (ClonExpress II One Step Cloning Kit, Vazyme, China), resulting in the pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 plasmid (BBa_K5507010). The resulting colonies were screened for ampicillin resistance and confirmed by PCR and DNA sequencing of the amplified DNA fragment (Figure 2B, 2C).

b. Construction of transgene K. xylinus-gdh KO

The plasmids pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 (BBa_K5507010) was introduced into K. xylinus, located at the gdh gene loci via electroporation. The resulting gdh knockout mutant (gdh KO) was screened for kanamycin resistance and confirmed by PCR (Figures 3).

c. Growth curves of K. xylinus-gdh KO

First of all, we wanted to make sure that our engineered K. xylinus-gdh KO can still grow normally. The 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 knockout gdh did not dramatically influence cell metabolites (figure 4).

d. Bacterial cellulose production assay

Then, we proceeded to test bacterial cellulose production. The wild-type K. xylinus and the gdh KO 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 bacterial cellulose. After 7 days, following a simple purification process, the bacterial cellulose yield in the media was measured.

The results confirmed that the WT K. xylinus can produce a relativity small amount of BC. The knockout of gdh gene increased bacteria cellulose (BC) production by about 52%. Compared to single mutant, pgi-OE gdh-KO showed massive BC overproduction, increased contents by 227% (Figure 5).

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 bacterial cellulose (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.

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 6). Therefore, the application of BC during tree planting helps to slow soil moisture loss.