Using plasmids as vectors to introduce foreign genes into engineered bacteria is the most common approach in genetic engineering. But plasmids in microbiology is unstable, which is major caused by genetic instability and structural instability:

(1) Structural instability: Generally speaking, foreign genes for engineered bacteria are not necessary, which means when the foreign genes mutant and loss their function, the host can still survive, so the mutations would gradually accumulate and cause great harm to foreign genes. Meanwhile, because plasmids could exist in many forms, some host use homologous recombinases to integrate plasmids, resulting in damage to the plasmids’ structure.

(2) Genetic instability: when the engineered bacteria divide, there is usually no mechanism to guarantee the average distribution of plasmids, so foreign genes imported through plasmids do not have genetic stability.

The degradation of engineered bacteria is essentially caused by growth rate. Degraded strains have more growth advantages than the non-degraded ones. Because plasmids itself will bring additional metabolic burden for engineered bacteria or the expression of foreign genes have some toxicity, therefore the more copy of the plasmids or the more expression of the toxic protein, bacteria grow slower. On the contrary, the small copy number, no plasmid or plasmids loss of function, the bacteria’s growth rate is much higher than that of non-degraded strains. Eventually, in the whole population of bacteria, degradation strains accumulate more and more, and functional strains less and less.

Currently, the conventional method is to add an independently expressing resistant gene into the plasmids. The resistant gene can degrade a special kind of antibiotic, so the bacteria with plasmids can obtain resistance to the antibiotic. Bacteria with low or no copies of plasmids will be killed or suppressed when antibiotics were added into the culture, while strains with high copies retained. This method is widely used in the laboratory. However, there are some disadvantages:

(1) the problem of structural instability of plasmids cannot be solved. After the target gene loses its function, the resistant gene can still be expressed smoothly, which leads to that the bacteria screened by the resistant environment are not thoroughly.

(2) the application of this method is restricted. It could be used in the laboratory, but there would be a huge cost due to the using of antibiotics in industry;

(3) those bacteria with resistant genes will metabolize antibiotics, so the concentration of antibiotics in the culture would gradually reduce, which is not conducive to long-term experiments.

(4) adding antibiotics to the culture is exactly adding selection pressure, so engineered bacteria need to use a large part of energy to fight against those survival pressure, which leads to relatively low expression of target genes in engineered bacteria.

To sum up, the use of antibiotics in the construction, screening and application of engineered bacteria can NOT be avoided, but there are also many problems. In this summer, BNU-china are devoted to explore a novel method to fight against plasmids loss in the environment without antibiotics, and hope that our efforts could finally change the world.

The great commercial success of Paclitaxel and other anti-microtubule medicines has inspired pharmaceutical companies to extract and test similar compounds, farmers to grow related plants. So an effective method is being needed urgently to discover many other similar compounds. Moreover, testing the concentration of paclitaxel from fermentation broths or plants are in high demand.

How to test the taxol and screening other compounds?
We determine to use microtubule for assisting.

As we all know, the mechanism of taxol is to kill cancer cells by obstructing the function of microtubule and consequently blocking cell division. Microtubules are a kind of important cellular structure composed of two monomers: α-tubulin and β-tubulin. These hollow rod shaped proteins are required for many cellular activities including cell division and transportation.^[2] A dynamic equivalence are found in microtubules, meaning that protein monomers are assembling and disassembling at every moment. The anti-microtubule agents can destroy the dynamic balance in microtubules, hence terminating cell mitosis and inducing the tumor cell apoptosis.

There are two types of anti-microtubule agents. One type inhibits assembly, such as vinca alkaloids, colchicine, podophyllotoxin and etc. The other type interferes disassembly, like taxanes and epothilones.

As for the discovery of anti-cancer compounds, we narrow down our sight to the anti-microtubule agents which are of great significance in cancer treatments.

As for our project this year, we modified the homo sapiens α-tubulin, ligated it with N/C terminal of the luciferase report gene fragments. Based on the principles of synthetic biology, we aimed to express the fusion proteins with α-tubulin and signaling residues. Then we made a kit containing the fusion α-tubulins and non-fusion β-tubulins with buffer which has an appropriate condition verified by experiments. We call the kit “taxolight”, and through which we can achieve these things below:

Screening with high feasibility

Anti-cancer agents especially paclitaxel have showed their powerful ability in clinical application. However, we still need to look for new drugs that are more effective.

The existing screening method of anti-microtubule agents needs to purify tubulins coming from mammalian brains. It heavily relies on the turbidity of tubulin solutions when they aggregate or disaggregate under certain temperatures in vitro. Once using this method, we can get a “S”-type standard aggregation curve based on the liquid OD value and the incubation time. Similarly, we can also get a standard disaggregation curve. When added different anti-microtubule agents, the aggregation/disaggregation curve will change correspondingly. Thus we can determine the role of the drug according to the change of curve.

The defects of this method are shown below:

1. The operation of extracting and purifying tubulin from animal brain is very complicated, and the experiment must be done within an hour after killing the animal. At the same time, the price of reagents in this experiment is expensive. The experiment period is long which takes no less than 3 days.
2. The wave length of measuring OD is 350nm, which is between ultraviolet light and visible light and always leads to a huge deviation. Also, the requirement of the testing instruments is high, quartz containers are needed as well, which cost a lot.

Our project avoids these drawbacks, and provides a new insight for the anti-microtubule drug screening. What we need is just a fluorescence microscope by using our kit.

Take paclitaxel as a control, we could test the fluorescence intensity of new medicine compared with paclitaxel's. In this way, further research and development of new anti-microtubule agents can be carried out easily than before.

Detection in high sensitivity

HPLC/RP-HPLC is one of the most common method to detect paclitaxel now. It relies on pumps to pass the sample through a column filled with solid adsorbent materials. Each component in the sample interacts differently with the adsorbent material, causing different flow rates and leading to the separation of the components as they flow out of the column.

This is a time-consuming process which is very unfavorable to the studies in laboratory. For example, in a laboratory which producing paclitaxel from fungus, detecting the concentration of the product may delay experiment process if there were no paclitaxel at all. So we need to develop an effective method to rapidly detect whether taxanes exist or not before measuring concentration. Our kit can reach the goal in order to accelerate research progresses.

Concentration detection

Apart from our former achievements, optimization is also needed. An intensity-concentration database of certain medicines (e.g. paclitaxel) is being planned, then we can use our “taxolight” to determine the concentration of this certain medicine conveniently.

There is a limited issue that the sample solution must be ensured not containing other anti-microtubule agents. Nevertheless, it is still useful. For example, farmers who plant taxus can apply our kit to detect the concentration of taxanes in there plants. Moreover, factory can use our kit to test the concentration of taxol in their fermentation broth. In conclusion, our product can be popularized in many agent-specific tests.