Difference between revisions of "Part:BBa K1017202:Experinece"

(Created page with "====37°C RBS==== <p>Using biobrick, P<sub>cons</sub> + 37°C RBS + mGFP+J61048, we tested the function of the 37°C RBS at room temperature (around 25°C) and at 37°C.</p> [[Fi...")
 
 
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====37°C RBS====
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====rRBS efficiency====
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<p>Using biobrick, P<sub>cons</sub> + 37°C RBS + mGFP+J61048, we tested the function of the 37°C RBS at room temperature (around 25°C) and at 37°C.</p>
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<p>We used the following biobrick to test the translational efficiency of K1017202 (rRBS) compared to the efficiency of other RBSs:
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[[File:NCTU_37RBS_biobrick.png|center|400px|Figure 1. The biobrick for testing the function of 37°C RBS.]]
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#P<sub>cons</sub> + '''BBa_B0034''' + mRFP + Ter
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[[File:NCTU_Test_37RBS_fluo_pic.png|center|600px|Figure 2. The differences between the level of GFP expressions can be easily observed under UV light.]]
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#P<sub>cons</sub> + '''[https://parts.igem.org/wiki/index.php?title=Part:BBa_K1017202 BBa_K1017202]'''+mRFP+Ter
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[[File:NCTU_Test_37RBS_ependorf.png|center|600px|Figure 3. The differences between the level of GFP expressions.]]
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#P<sub>cons</sub> + '''BBa_B0030''' + mRFP + Ter
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#P<sub>cons</sub> + '''BBa_B0032''' + mRFP + Ter
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#control: pet 30
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</p>
  
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As shown in Figure 4 the normalized expression, obtained by dividing the florescence expression (emmision 612 nm and excitation 584 nm) with the OD<sub>600</sub> value, of GFP under 37°C is much higher than the expression under room temperature. Such result demonstrates the fact that 37°C RBS can effectively regulate gene expression by responding to temperature. The increased kinetic energy at 37°C is sufficient to cause the 37°C RBS to unfold and become available for ribosome binding. At room temperature, however, there isn't sufficient kinetic energy to unfold the hairpin structure and the structure is preserved. As a result, the translational efficiency is very low at room tempersture.
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<p> As you can see from Figure 5, Figure 6, and Figure 7, the bacterial pellet and liquid of each biobrick shows different level of 'RFP expressions as the RBS of each biobrick provides a different translation efficiency. The deeper the red color is, the higher the level of expression is. </p>
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[[File:NCTU_Test_RBS_pic.png|center|600px|Figure 5. B0034 clearly expressed more ''RFP'' compared to others.]]
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[[File:NCTU_Test_RBS_flo_pic.png|center|600px|Figure 6. K1017202 shows moderate RFP expression that can be distinguied from the expressions B0030 and B0032 ]]
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[[File:NCTU_Test_RBS_ependorf.png|center|600px|Figure 7. From left to right, the ribosome biding sites respectively: B0032, K1017202, B0030, B0034, control.]]
  
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[[File:NCTU_Test_functional_test_of_37RBS.png|center|600px|Figure 4. The normalized expression (florescence expression/ OD value) under 37°C is higher than the expression under room temperature by about 6 folds.]]
 
  
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<p> We measured the normalized expression for each biobrick mentioned above. The result is shown in Figure 8. We calculated the normalized expression by dividing fluorescence expression with the OD value measured, since the higher the OD value is, the larger the amount of bacteria that can express florescence would be. As shown in Figure 8, the normalized expression of the biobrick with B0034 is the highest and the one with  K1017202 (rRBS) is the second highest, while the other two of B0032 and B0030 show  weak expressions. This result implies that K1017202 can, in fact, serve as a functional RBS. In comparison to other RBS, K1017202 can provide moderate translational efficiency that is just lower than that of the highly efficient B0034. </p>
  
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[[File:NCTU_Test_functional_test_of_different_RBS.png|center|600px|Figure 8. K1017202 shows moderate expression compared to the others.]]
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Latest revision as of 18:40, 27 September 2013

rRBS efficiency

We used the following biobrick to test the translational efficiency of K1017202 (rRBS) compared to the efficiency of other RBSs:

  1. Pcons + BBa_B0034 + mRFP + Ter
  2. Pcons + BBa_K1017202+mRFP+Ter
  3. Pcons + BBa_B0030 + mRFP + Ter
  4. Pcons + BBa_B0032 + mRFP + Ter
  5. control: pet 30

As you can see from Figure 5, Figure 6, and Figure 7, the bacterial pellet and liquid of each biobrick shows different level of 'RFP expressions as the RBS of each biobrick provides a different translation efficiency. The deeper the red color is, the higher the level of expression is.

Figure 5. B0034 clearly expressed more RFP compared to others.
Figure 6. K1017202 shows moderate RFP expression that can be distinguied from the expressions B0030 and B0032
Figure 7. From left to right, the ribosome biding sites respectively: B0032, K1017202, B0030, B0034, control.


We measured the normalized expression for each biobrick mentioned above. The result is shown in Figure 8. We calculated the normalized expression by dividing fluorescence expression with the OD value measured, since the higher the OD value is, the larger the amount of bacteria that can express florescence would be. As shown in Figure 8, the normalized expression of the biobrick with B0034 is the highest and the one with K1017202 (rRBS) is the second highest, while the other two of B0032 and B0030 show weak expressions. This result implies that K1017202 can, in fact, serve as a functional RBS. In comparison to other RBS, K1017202 can provide moderate translational efficiency that is just lower than that of the highly efficient B0034.

Figure 8. K1017202 shows moderate expression compared to the others.