PCR fragment (lumbrokinase gene) insertion
Blue-white Screening

After engineering our parts in E.coli DH5α, our team utilized blue-white screening to detect the insertion of the PCR fragment. In anticipation, the PCR fragment (cDNA of Lumbrokinase) should be successfully inserted into the lacZ gene in pGEM-T EASY vector, thereby resulting in a functional gene for the subsequent steps. In the experiment, we added IPTG as an inducer to induce the expression of lacZ gene; it allows us to identify the successfully ligated plasmid in E.coli DH5α and the failed ones. If the PCR fragment wasn’t inserted into the lacZ gene properly, X-gal will be decomposed and produce blue colonies.

In our experimental results, we observed particular colonies exhibiting a white coloration instead of blue, which suggested that the lacZ gene did not express normally because the multiple cloning site (MCS) on the lacZ gene was occupied by specific substances (see figure 2 and 3) Therefore, it can be proven that there was foreign DNA inserted into the lacZ gene.

Small scale restriction enzymatic check

In figure 4, we can see the differences between the length of each sample. By analyzing the result, it can be indicated that the clones in sample 3 and 4 are considered to contain pGEM-T EASY vector that is only cleaved at the Ncol site with no DNA fragment inserted since the bands of the two samples exhibited the complete 3kb length of the pGEM-T EASY vector. In comparison, the bands of sample 1, 2, 5, and 6 exhibited bands with longer length that are closer to the wells, revealing the successful insertion.

However, since the first band exhibits shorter length than the second and sixth band, there are still differences between the band of sample 1 and the bands of sample 2 and 6. As seen in figure 5, the location of the restriction enzyme sites affect whether the fragments are connected forward or reversely. From this, our team concluded that the first band is the pGEM-T EASY vector with DNA fragment connected reversely, while the second and the sixth band are the pGEM-T EASY vector with DNA fragment connected forwardly. Therefore, samples 1, 2, and 6 were once again proven of having successful DNA fragment insertion.

M 1 2 3 4 5 6

To see more detailed information, please visit the experimental procedure and result page in the wet lab.

The Lumbrokinase gene within E.coli DH5α DNA sequencing

Our team also conducted DNA sequencing to confirm whether the Lumbrokinase gene had been successfully ligated with the pGEM-T EASY vector since the results of DNA sequencing offers us the exact sequence of nucleotides or bases of the DNA molecule in our plasmid. Figure 6 demonstrates the result and the following features of our pGEM-T EASY vector.

In the first line within the red square, the number of the identities compares the percentage of the gene on plasmid that completely matches with the DNA sequence of Lumbrokinase. The identity number of our result is 744/754 (99%), indicating a strong alignment between the DNA sequences in our experimental clones and the lumbrokinase gene. Moreover, the DNA sequences highlighted with yellow are the sequences of the lumbrokinase, while the unhighlighted ones beneath are the DNA sequences of the DNA fragment we inserted into the vector. Upon comparing the two sequences, it becomes evident that they exhibit a high degree of similarity. Based on the above evidence, the gene fragment of lumbrokinase was proven to be successfully ligated with the pGEM-T EASY vector in our experiment.

More information and detailed results, such as the number of max score, total score, and E value, can be found in the result page

IPTG Induction

By means of enhancing traditional methods of industrial production, our team employed IPTG induction as a switch-like mechanism that allows the lumbrokinase production to be produced when E. coli BL21 receives a specific chemical: IPTG. IPTG is a molecular analog of allolactose that is capable of removing a repressor from the lac operon to induce gene expression, allowing the RNA polymerase to bind on the lac promoter and start .

Western-blotting

In the experiment, our team performed western-blotting as a final verification to confirm the production of lumbrokinase through IPTG induction. In figure 8, the major bands in the result of 2h, 4h, 6h, and 8h exhibited lumbrokinase production with good quantity, revealing the effectiveness of IPTG induction. Also, the bands in the 6h result displayed the highest lumbrokinase production compared to the result in other periods of time, indicating that the accurate peak production time of IPTG-induced lumbrokinase occurred at 6h. Consequently, the effectiveness and efficiency of IPTG induction was proven to be successful, and the mechanism of IPTG induction can be applied to industrial production to improve the traditional production method of lumbrokinase.

Oleate Induction

In the experiments, our team designed a switch-like mechanism called oleate induction that could be applied to the probiotic platform due to its operating mechanism. Since the presence of fatty acids in blood vessels may aggravate white blood clots, our team takes oleate as a specific chemical to achieve the effect of induction and deactivation. In the experiment, we modified the pET-22b-LK vector designed for IPTG induction to the vector for oleate induction by replacing the lac operator with fdH operator (the fadR binding site).

When fatty acid is absent, fadR (repressor) will bind to the fadR binding site called fadH operator to block the operation of protein transcription. However, when fatty acid is present, it will be degraded into acetyl-CoA, which thereby releases the repressor fadR and allows RNA polymerase to bind on the operating site and start transcription to produce lumbrokinase.

Gel electrophoresis

In the experiment, our team used gel electrophoresis to confirm whether the primers annealed correctly after we modified the pET 22b vector. Since the annealed band hides the nitrogenous bases inside and exposes the phosphates outside, the structure tends to be negatively charged. In figure 9, the results show that the annealed band moves faster due to the charge difference compared to the forward, reverse and 100°C thermal annealed bands. Therefore, the primers were proved to be correctly annealed.\ though our experiment.

M F R Anneal Anneal+heat

In order to confirm the removal of lac operator from the pGEM-T EASY vector, our team conducted a gel analysis involving three samples: the uncut vector, the vector cleaved by BglII, and the cleaved by both BglII and XbaI. In the result of figure 10, since the lac operator has a very short length and was barely possible to be seen in the gel result, the removal of lac operator was proven to be successful as the third sample closely resembled the band from a single cut.

M uncut PC LK

Western-blotting

In the experiment, our team performed western-blotting as a final verification to confirm the production of lumbrokinase through oleate induction. In figure 11, the major bands in the result of 6h, 8h, 24h, and 8h exhibited the production of lumbrokinase, revealing that the oleate induction worked. Also, the bands in the 24h result displayed the highest lumbrokinase production compared to the result in other periods of time, indicating that the accurate peak production time of oleate-induced lumbrokinase occurred at 24h.

Additionally, our team assessed the productivity of oleate induction by adding varying concentrations of fatty acids. In figure 12, all of the samples demonstrated a similar yield in terms of lumbrokinase production. Upon analyzing the result, our team determined that lumbrokinase was able to be induced under a distinct yield of fatty acid, which suggested that lumbrokinase could be induced and remain high stability even with a 2.5 mM oleate concentration. Consequently, the oleate induction was proven by the above information to be effective and is able to be applied to the probiotic platform.

IPTG Induction

Absorption by Intestine Cells

In order to ensure the effective passage and absorption of lumbrokinase through intestinal cells, we have devised a method involving the detection of lumbrokinase levels in the bloodstream of mice that have ingested the probiotic platform. This method involves labeling the lumbrokinase produced by E.coli with a fluorescent protein, enabling us to identify and assess the quantity and trend of lumbrokinase absorption from the intestine into the blood vessels. If our team does observe the protein highlighted in the vessels, it means that lumbrokinase is successfully absorbed and goes through the intestine cells.

The Temperature and pH Measurement

With the sim of verifying the capacity and the period of time that the probiotic platforms are able to produce functional lumbrokinase in the human intestine, our team decided to measure the range of pH and temperature that is suitable for E.coli to live and produce Lumbrokinase functionally. By utilizing the collected and analyzed data, we can make a comparison between the human intestinal environment and the experiment results. If the results indicate that the optimal the pH and temperature ranges for E. coli probiotics to live in fit the environment of the human intestine condition, it will be possible that E.coli probiotics are able to stay in the human intestine and produce functional lumbrokinase safely for a long period.

Thought Mice Experiment - the function of lumbrokinase to blood clots

In order to ensure the effective action of lumbrokinase in dissolving blood clots within human vessels, we have devised an experiment involving the observation of blood clot conditions in mice after they have administered the probiotic platform. Figure 13 provides a visual representation of the concept and rough procedures in the mice experiment. Our team decided to verify whether the oleate induction and the probiotic platform in mice intestines works to affect the blood condition since they are the two main points to show the success of our project.

Firstly, we decided to separate the mice into two groups. The first group of mice are both on high fat diets; however, one mouse ingest the in-vivo probiotic platform in its intestine and another does not. The second group of mice both contain a probiotic platform in their intestines; however, one takes a high fat diet, and the other takes a control diet. We planned to compare the blood pressure, existence of fluorescent protein, and blood component to ensure the function of probiotic platform and lumbrokinase in dissolving blood clots.

During the upcoming mice experiment, we may consider adding fluorescent protein genes onto the vector to produce lumbrokinase with a fluorescent protein. This approach will make it easier to track the position of lumbrokinase in the vessels after lumbrokinase has been absorbed by the intestines .

If there is any improvement in the thrombus condition or reduction in the quantity of blood clots, it may suggest that not only was lumbrokinase successfully transported into the arteries but also that it functions in dissolving blood clots. The mice experiment enables us to assess the efficacy of lumbrokinase within a physiological context and its potential as a therapeutic agent for addressing blood clot-related issues.

Industrial production

Given our team's goal to enhance the efficiency of traditional lumbrokinase producing methods, we hope to identify the optimal temperature and pH ranges at which IPTG induction works the best and E. coli BL21 produces the most proteins, thereby maximizing the efficacy of the industrial production.