Difference between revisions of "Team:SSTi-SZGD/Results"

Fed-batch fermentation of recombinant hasA and HA production

A fed-batch fermentation strategy was employed to ensure an optimal HA production over a time course. We performed a 48 h high-cell-density fermentation experiment using MM medium with xylose as an inducer. Sucrose was fed to the batch at designated time points. HA concentrations and cell growth densities were measured every 12 h.

Figure 4: analysis of HA production level of recombinant hasA and cell growth. A typical time course of recombinant hasA expression in B. subitlis 168E during xylose induction phase. HA production (red, mg/L), and cell growth density (black, OD600 value) were measured at regular intervals.

The results showed that HA production increased at a steady rate as time passed, reaching over 300mg/L (CTAB method) at the 50 h; while bacterial cells maintained normal growth pattern and experienced lag phase dropping in the final hours. These results confirmed the success of HA production by recombinant B.subtilis 168E strain.

LHAase functional and expression level analysis

①Growth rate vs. LHAase expression level

Similar to HA production, a fed-batch fermentation strategy was also employed to ensure maximal expression of LHAase over a time course. We performed a 60 h high-cell-density fermentation experiment with feeding of fresh culture media every 12 h. LHAase production and cell growth density were measured at regular interval. As expected, fusion of the signal peptide and 6xHis tag resulted to a distinct accumulation of extracellular LHAase in the flask culture. (Figure 11) showed that LHAase expression increased exponentially as time passed, enzymatic activity up to 1.149*105 U/ml at the final 60 h (in pDG1730 vector); on the other hand, overexpression of LHAyal did not seem to affect normal bacterial growth (Figure 11). Therefore N-terminal engineering enables an extracellular secretion of hyaluronidase. A visual HA plate assay was also conducted to verify and confirm the activity of LHAase. HA hydrolysis by the crude culture supernatant resulted to the appearance of a circle zone around the hole, indicating that leech LHAase has distinct hyaluronidase function and can be successfully overexpressed in B. Subtilis (Figure 12).

Figure 11: analysis of enzyme activity of recombinant LHAase and cell growth. A typical time course of recombinant LHAase expression in B. subitlis 168. LHAase enzyme activity (red, U/ml), and cell growth density (black, OD600 value) were measured at regular intervals.

Figure12: Determination of LHAase activity using a HA plate assay, where cylindrical holes were injected with fermenting supernatant of LHAase-expression (pDG1730, pMA0911) or control (empty vector) B. subtilis 168. A positive control denoted to commercial BTH, while a negative control denoted to water. The white circles in pDG1730 and pMA0911 showing the color residues from fermenting culture.

②LHAase expression at transcriptional level

In addition, we extracted total RNA at each indicated time point to understand the expression pattern at transcriptional level. By reverse transcribing mRNA to cDNA and performed RT-PCR using LHAyal primers, the results showed that mRNA levels accumulated up to 48 h and seemed to drop at 60 h (Figure 13). Given Figure 11 showing that LHAase production continued to rise up to 60 h, it is possible to assume that LHAase production may cease to increase after 60 h, due to a drop in mRNA transcripts after 48 h of bacterial growth. In the future we will explore this further.

Figure13: Agarose gel electrophoresis of mRNA transcript obtained at various time points. (1-7): 6h, 12h, 24h, 36h, 48h, 60h, NTC. Top strand showing RT-PCR products amplifying a section of LHAyal coding sequence, bottom strand showing RT-PCR products amplifying a housekeeping gene sequence (16s rRNA).

③LHAase purification and analysis

LHAase was then purified from crude supernatant sample using a Ni-NTA affinity column and analyzed by SDS-PAGE. A clear band was shown with a molecular weight approximately 58 kDa (Figure 14), which is consistent with the value published in previous research (3).

Figure 14: SDS-PAGE analysis of LHAase in total extracellular crude protein fraction from recombinant B.subtilis harboring pMA0911-AmyX-H6LHAyal. A 12% SDS-PAGE gel was used. Lane 1: purified LHAase by Ni-NAT affinity column; Lane 2: the crude extracellular protein fraction

Fabrication of HA-cHA Micro-needles

In order to successfully fabricate a HA microneedle, we tested different weight ratio of the HA molecules and cHA. The experimental results showed that the ratio of HA-cHA (0:1) forms a lump after overnight curing and is easily broken into powder. The ratio of HA-cHA (1:1) is difficult to be demolded after overnight curing, and could not form a complete needle shape, while being poor in mechanical properties and spreadability. The ratio of HA-cHA (5:1) forms complete microneedles after overnight curing, and the patch is easily to be demolded, with high mechanical strength and spreadability (Figure 20). Therefore, the ratio of HA-cHA (5:1) is the most suitable ratio for preparing microneedles that have appear to be strong and sharp enough to penetrate into the skin. It took less than 48 h to complete a microneedle patch and the required materials are minimal. Microneedle made from micromold-based fabrication technology is suitable for mass production due to high production efficiency, and repeat-using of the mold.

Figure 20: HA microneedle fabrication. (a) micromold is filled with HA-cHA homogenous suspension; (b) demolded after curing overnight; (c-e) display of needles made of different weight ratios of HA and cHA.