Difference between revisions of "Team:Wageningen UR/ecoli survival"
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| + | </comic><section id="MMlb_intro"> | ||
| + | <h1 text-align='center'>Testing chassis survival in sugar water</h1> <!-- Written as abstract--> | ||
| + | <p>The final BeeT product will be applied using sugar water, which is transported into the hive by worker bees. This means that our chassis needs to be able to survive in a nutrient limited environment, which does have a high sucrose content. In order to test this, we simulated a sugar water environment and tested whether bacteria can survive it. The result of this was that our chassis bacteria, <i>Escherichia coli </i>, was able to survive up to at least an entire day in the sugar water. This means that the bees will have at least 24 hours to pick up some of our BeeT.</p> | ||
| + | <!-- "Why did we start this experiment"--> | ||
| + | <p>The modeling results from the <a href=https://2016.igem.org/Team:Wageningen_UR/Model#metabolic > metabolic modeling </a> project only showed what would happen in the time period of 90 seconds to 90 minutes after being put in the sugar water. Because of this, more evidence was needed to see what would happen to the <i>E. coli </i> in the sugar water for the time scales we are interested in. We know the presence of the high amount of sugar water leads to osmotic stress.<sup><a href="#mm1" id="refmm1">1</a></sup> | ||
| + | This is because the sucrose molecules from the medium are not able to travel across the cell-membrane, but still pull the water molecules present inside the cell towards them. The effects of the osmotic stress on the bacterium were not clear outside of the 90 minute range determined by the metabolic model. However, as determined by the <a href=https://2016.igem.org/Team:Wageningen_UR/Model#beehave>BEEHAVE model</a>, we require that the BeeT survives for days in the sugar water. In this project we aimed to experimentally determine how long BeeT can survive in osmotically unfavourable conditions. </p> | ||
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| + | <section id="MMlb_results"> | ||
| + | <h2>Testing Survival </h2> | ||
| + | <p> The results from the experiments were a great success, as you can see in Figure 1 and 2. The result of experiment 3 is that the <i>E. coli</i> was still able in the LB was still possible after the 24 hours in the sugar water solution. | ||
| + | All three experiments clearly show that sugar water is not as lethal as first hypothesized. Not only that, but the results fit with the model hypothesis in that long-term survival is indeed possible. Thus we have shown that <i> E. coli</i> can indeed be used as a chassis for our BeeT. This result was taken into account in the <a href=https://2016.igem.org/Team:Wageningen_UR/Model#beehave>BEEHAVE model</a>. | ||
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| − | <img src="https://static.igem.org/mediawiki/2016/ | + | <img height="301" width="500" src="https://static.igem.org/mediawiki/2016/2/2f/T--Wageningen_UR--ecoli_exp1_success.jpg"> |
| + | <figcaption>Figure 1. Photo of plate 2 of the repeat of the first experiment. The top row shows growth after having spent 30 minutes in the sugar water and then overnight growth. The time step increases for row 3 to 50 minutes, row 5 to 90 minutes, and row 7 to 120 minutes. Row 8 and all intermediate rows are negative controls. The first three columns are replicates of 0g/L sucrose, then 3 columns of 100g/L sucrose, then 3 columns of 200g/L sucrose, and finally 3 columns of 400g/L sucrose. (The image appears to be flipped upside down for some reason, in case it shows as such, the pencil marks are in the upper right corner of the plate)</figcaption> | ||
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| − | + | <img height="300" width="500" src="https://static.igem.org/mediawiki/2016/5/58/T--Wageningen_UR--ecoli_exp2_success.jpg"> | |
| − | + | <figcaption>Figure 2. Photo of plate 2 of the second experiment. The top row shows growth after having spent 1 hour in the sugar water and then overnight growth. The time step increases for row 3 to 2 hours, etc (as shown in the right side of the photo). Row 8 and all intermediate rows are negative controls.The first 4 columns are replicates of 625g/L sucrose concentration, whilst the last 4 columns are replicates of the 312.5g/L sucrose concentration conditions. </figcaption> | |
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| − | + | </p></section> | |
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<section id="MMlb_mm"> | <section id="MMlb_mm"> | ||
| − | <h2> | + | <h2>Method of testing</h2> |
| − | <p> The method used to test the survival of <i>E. coli</i> in sugar water | + | <p> The method used to test the survival of <i>E. coli</i> in sugar water is a <a href=2016.igem.org/Team:Wageningen_UR/Experiments#ecoli>simple growth experiment</a>. First, the bacteria grow overnight in LB medium. Then, 5 µL per sample of this is pipetted into various concentrations of sterile sugar water made of varying amounts of sucrose, ranging from 0 g/L up to 625 g/L, and sterilized tap water. We then took 5 µL samples of the sugar water and inoculated them into fresh 200µL LB medium wells and left to grow overnight. </p> |
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| − | <section id=" | + | <section id="MMlb_refs"> |
| − | <h2> | + | <h2>References</h2> |
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| − | + | <a id="mm1" href= https://www.microbiology.ubc.ca/sites/default/files/roles/drupal_ungrad/JEMI/15/JEMI15_15-21.pdf >1.</a> | |
| + | Cheng, Y. L., Hwang, J., & Liu, L. (2011). The Effect of Sucrose-induced Osmotic Stress on the Intracellular Level of cAMP in Escherichia coli using Lac Operon as an Indicator. Journal of Experimental Microbiology and Immunology (JEMI) Vol, 15, 15-21. | ||
| + | <a href="#refmm1" title="Jump back to footnote 1 in the text.">↩</a> | ||
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Latest revision as of 16:07, 18 October 2016
Testing chassis survival in sugar water
The final BeeT product will be applied using sugar water, which is transported into the hive by worker bees. This means that our chassis needs to be able to survive in a nutrient limited environment, which does have a high sucrose content. In order to test this, we simulated a sugar water environment and tested whether bacteria can survive it. The result of this was that our chassis bacteria, Escherichia coli , was able to survive up to at least an entire day in the sugar water. This means that the bees will have at least 24 hours to pick up some of our BeeT.
The modeling results from the metabolic modeling project only showed what would happen in the time period of 90 seconds to 90 minutes after being put in the sugar water. Because of this, more evidence was needed to see what would happen to the E. coli in the sugar water for the time scales we are interested in. We know the presence of the high amount of sugar water leads to osmotic stress.1 This is because the sucrose molecules from the medium are not able to travel across the cell-membrane, but still pull the water molecules present inside the cell towards them. The effects of the osmotic stress on the bacterium were not clear outside of the 90 minute range determined by the metabolic model. However, as determined by the BEEHAVE model, we require that the BeeT survives for days in the sugar water. In this project we aimed to experimentally determine how long BeeT can survive in osmotically unfavourable conditions.
Testing Survival
The results from the experiments were a great success, as you can see in Figure 1 and 2. The result of experiment 3 is that the E. coli was still able in the LB was still possible after the 24 hours in the sugar water solution.
All three experiments clearly show that sugar water is not as lethal as first hypothesized. Not only that, but the results fit with the model hypothesis in that long-term survival is indeed possible. Thus we have shown that E. coli can indeed be used as a chassis for our BeeT. This result was taken into account in the BEEHAVE model.
Method of testing
The method used to test the survival of E. coli in sugar water is a simple growth experiment. First, the bacteria grow overnight in LB medium. Then, 5 µL per sample of this is pipetted into various concentrations of sterile sugar water made of varying amounts of sucrose, ranging from 0 g/L up to 625 g/L, and sterilized tap water. We then took 5 µL samples of the sugar water and inoculated them into fresh 200µL LB medium wells and left to grow overnight.
References
