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| | <article id="project" class="col-lg-10 col-lg-offset-1 col-md-12 col-md-offset-0 col-sm-offset-0 col-sm-12"> | | <article id="project" class="col-lg-10 col-lg-offset-1 col-md-12 col-md-offset-0 col-sm-offset-0 col-sm-12"> |
| | <header class="page-header"> | | <header class="page-header"> |
| − | <h1>Background</h1> | + | <h1>Demonstration</h1> |
| | </header> | | </header> |
| − | | + | <h2>1.</h2> |
| − | <p>Using plasmids as vectors to introduce foreign genes into engineered bacteria is the most common approach in genetic engineering. But plasmids in microbiology are unstable, which is major due to genetic instability and structural instability:</p> | + | <p>We have designed the basic functional parts including gdh, cI protein, emrR protein and emrR binding promoter. Gdh and cI protein is well character by using SDS-Page gel electrophoresis and western blot. These parts can worked well separately in E.coli when cultivated in LB medium containing appropriate amount of sAmp antibiotics and IPTG inducer.</p> |
| − | <p>(1) Structural instability: Generally, foreign genes are not necessary for engineered bacteria, which means whether the foreign genes are functional, the host can still survive, so the unfavorable mutations would gradually accumulate leading to strain degeneration. Meanwhile, plasmids in bacteria could be in many forms. Some can be integrated into genomes of the host by homologous recombinases.</p> | + | <p>In our project, gdh works as a growth factor that successfully accelerated the growth of bacteria, which is clearly showed in figure1A. Gdh is a 89kDa protein and in our design, we added V5-tag to the C-terminus of the coding sequence, so we can process the SDS-Page gel electrophoresis and western blot (figure1B) to make sure it is gdh that cause the growth acceleration. </p> |
| − | <p>(2) Genetic instability: there is usually no mechanism to guarantee the average distribution of plasmids during division, so the genes in exogenous plasmids do not have genetic stability.</p> | + | |
| − | <p>The degradation of engineered bacteria is essentially caused by growth rate. Degraded strains have more growth advantages than the non-degraded ones because of the additional metabolic burden taken by plasmids or the toxic substance expressed by foreign genes.Therefore the more copies of the plasmids or the higher expression of the toxic protein, bacteria grow slower. On the contrary, the bacteria with low copy number, no plasmid or whose plasmids loss functiongrow faster 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.</p>
| + | |
| | <figure class="text-center"> | | <figure class="text-center"> |
| − | <img src="https://static.igem.org/mediawiki/2018/2/2c/T--BNU-China--Degradation_of_engineered_bacteria.png" width="65%"> | + | <img src="https://static.igem.org/mediawiki/2018/8/86/T--BNU-China--image_d1.jpg" width="65%"> |
| | <figcaption> | | <figcaption> |
| − | Fig.1 Degradation of engineered bacteria | + | Fig.1 Demonstrations of gdh.cI protein is a 47kDa protein with V5-tag in C-terminus. We also use the electrophoresis and western blot to insure the expression of cI (figure2). |
| | </figcaption> | | </figcaption> |
| | </figure> | | </figure> |
| − | <p>Currently, the conventional method is to add a resistant gene expresses independently into the plasmids. 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:</p> | + | <figure class="text-center"> |
| − | <p>(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.</p>
| + | <img src="https://static.igem.org/mediawiki/2018/2/25/T--BNU-China--image_d2.jpg" width="65%"> |
| − | <p>(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;</p> | + | <figcaption> |
| − | <p>(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.</p> | + | Fig.2 Demonstrations of cI. |
| − | <p>(4) Adding antibiotics to the culture is exactly adding selection pressure, so a large part of energy is use to fight against those survival pressure, which leads to relatively low expression of target genes in engineered bacteria.</p>
| + | </figcaption> |
| − | <p>To sum up, the use of antibiotics in the construction, screening and application of engineered bacteria can NOT be avoided, and 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.</p>
| + | </figure> |
| | + | <p>Besides, we created the loop described in Figure 3a to test the function of emrR protein and its binding promoter. Bacteria is induced by SA. By monitoring the fluorescent density and its ratio to the OD value in the bacteria (Figure3), we confirm that emrR protein and its binding promoter can correctly work and have the potential to be used in integrated plasmids.</p> |
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