Difference between revisions of "Team:KUAS Korea/Results"
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| − | <ol | + | <ol><br>Cooperator expresses beta-glucosidase with constitutive promoter to degrade cellobiose around it. We had the cell stock of cooperator with pCEL vector containing beta-glucosidase and transformed it into E.coli BW25113. The medium plate contains cellobiose instead of glucose to check whether E.coli that we transformed expresses beta-glucosidase on its surface. The streaking side below shows that cooperator grows on cellobiose containing M9 minimal medium. |
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<br>[Fig 2.] Cultured cheater on M9 minimal medium with 0.48% glucose | <br>[Fig 2.] Cultured cheater on M9 minimal medium with 0.48% glucose | ||
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<img src="https://static.igem.org/mediawiki/2018/1/1b/T--KUAS_Korea--GFP_PART2.png" height="300px" style="float: center;" hspace="5" vspace="100"> | <img src="https://static.igem.org/mediawiki/2018/1/1b/T--KUAS_Korea--GFP_PART2.png" height="300px" style="float: center;" hspace="5" vspace="100"> | ||
<br>[Fig 2-1.] Cultured cheater under fluorescence microscopy showing the GFP activity | <br>[Fig 2-1.] Cultured cheater under fluorescence microscopy showing the GFP activity | ||
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| + | <header class="section-header"> | ||
| + | <h4><strong>3. Co-culture</strong></h4> | ||
| + | <div style="text-align:justify"> | ||
| + | <ol>As the cheater and cooperator reached OD600 1.8 in M9 minimal medium with glucose concentration 0.48%, we began co-culture in M9 minimal medium with cellobiose concentration 0.48%. Batch co-culture was conducted in 5ml of medium in ratio of cheater and cooperator 1:1, 1:2, 1:4, 2:1, 4:1. 1 was counted as 1ul and each medium was incubated for 1, 3 and 4 days. After each day, we streaked 100ul of the incubated medium on LB agar plate with ampicillin. However, expected bacteria did not grow at all after overnight incubation. | ||
| + | </ol><br> | ||
| + | <ol>We predicted possible causes of unsuccessful co-culture as follows; <ul> | ||
| + | <li>Too little amount of bacteria inoculated in the medium</li> | ||
| + | <li>Low survival rate of pre-cultured bacteria since the optimal OD600 was 1.0 and ours was 1.8 which is thought to be after stationary phase.</li> | ||
| + | <li>Too short period of incubation</li><br> | ||
| + | We plan to conduct the experiment once again with newly grown bacteria. | ||
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Latest revision as of 00:52, 18 October 2018
Results |
1. Beta-glucosidase expression
Cooperator expresses beta-glucosidase with constitutive promoter to degrade cellobiose around it. We had the cell stock of cooperator with pCEL vector containing beta-glucosidase and transformed it into E.coli BW25113. The medium plate contains cellobiose instead of glucose to check whether E.coli that we transformed expresses beta-glucosidase on its surface. The streaking side below shows that cooperator grows on cellobiose containing M9 minimal medium.
[Fig 1.] Cultured cooperator on M9 minimal medium with 0.48% cellobiose
2. GFP expression
- Cheater expresses GFP when it obtains glucose. We had the cell stock of cheater with the vector encoding GFP as a reporter gene and transformed it firstly into E.coli DH5α and then to E.coli BW25113.
[Fig 2.] Cultured cheater on M9 minimal medium with 0.48% glucose
[Fig 2-1.] Cultured cheater under fluorescence microscopy showing the GFP activity
3. Co-culture
- As the cheater and cooperator reached OD600 1.8 in M9 minimal medium with glucose concentration 0.48%, we began co-culture in M9 minimal medium with cellobiose concentration 0.48%. Batch co-culture was conducted in 5ml of medium in ratio of cheater and cooperator 1:1, 1:2, 1:4, 2:1, 4:1. 1 was counted as 1ul and each medium was incubated for 1, 3 and 4 days. After each day, we streaked 100ul of the incubated medium on LB agar plate with ampicillin. However, expected bacteria did not grow at all after overnight incubation.
- We predicted possible causes of unsuccessful co-culture as follows;
- Too little amount of bacteria inoculated in the medium
- Low survival rate of pre-cultured bacteria since the optimal OD600 was 1.0 and ours was 1.8 which is thought to be after stationary phase.
- Too short period of incubation
We plan to conduct the experiment once again with newly grown bacteria.
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