Difference between revisions of "Part:BBa K3763000"

Revision as of 16:30, 18 October 2021

RBS improved on the basis of BBa_B0030

Overview

The ribosome binding site (RBS) is a short sequence upstream the transcription start which is crucial for protein translation.
It has been proveed that its effectiveness is determined by its base-pairing potential to the ribosome and its spacing from the start codon. Therefore, under the condition that the latter remains unchanged, we believe that the function of RBS can be changed by changing its sequence

Characterization

Parts BBa_B0030 is a strong RBS based on Ron Weiss thesis. And its strength is considered relative to BBa_B0031, BBa_B0032, BBa_B0033 and BBa_B0034 by the former iGEM teams.

After consulting a large number of RBS data, we found that most RBS with higher binding efficiency had the repetitive sequence of "aggg", or "aaa" after the start codon. B0030 was the one with the highest expression efficiency among the four existing RBS, with the sequence of "aaa" but no sequence of "aggg". Therefore, we modified the sequence to change "aagagg" to "aggagg", so that it could simultaneously have the characteristics of two sequences with high binding efficiency. In later experiments, we wanted to compare the performance of the two sequences.

Figure 1. The change in sequence.

Figure 2. Plasmid design.

We further combine the two RBS with the promoter pFadD_Lac which is sensitive to fatty-acids. After the successful transformation of the plasmid, we used different concentrations of fatty acids for induction for different times. The expression level of eGFP was determined by fluorescence detection with a microplate reader, reflecting the performance of the RBS. The results are as follows:

Figure 3. BBa_K3763002(pFadD_Lac-RBS BBa_B0030-GFP-rrnB T1 and T2 terminator, pDSW208-99-2) contains RBS without modification.

Figure 4. BBa_K3763003(pFadD_Lac-RBS BBa_K3763000-GFP-rrnB T1 and T2 terminator, pDSW208-99-2) Contains RBS with modification

According to the results, we can find that the performance of our fatty acids-sensitive promoter is improved to a certain extent. Taking 0.125× fatty acid concentration as an example, the fluorescence intensity of 99-1 is over 11000 after 6 hours, while the promoter of 99-2 is less than 10000 at three fatty acid concentrations. However, we can see that the optimized promoter still has a high expression leakage and low expression in a short time to some extinct which is closely to the function of the promoter.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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-                  <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/b/b0/T--WHU_China--00_3_Results1.png" width="70%">
+                  <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/0/0c/T--WHU_China--00_4_Results2.png width="70%">
                   <p style="color:Gray; padding:0px 30px 10px;">Figure 3. BBa_K3763002(pFadD_Lac-RBS BBa_B0030-GFP-rrnB T1 and T2 terminator, pDSW208-99-2) contains RBS without modification.</p>
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          <div class="col-lg" style="margin:auto;text-align:center;">
-                  <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/0/0c/T--WHU_China--00_4_Results2.png" width="80%">
+                  <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/b/b0/T--WHU_China--00_3_Results1.png" width="80%">
                   <p style="color:Gray; padding:0px 30px 10px;">Figure 4. BBa_K3763003(pFadD_Lac-RBS BBa_K3763000-GFP-rrnB T1 and T2 terminator, pDSW208-99-2) Contains RBS with modification</p>
           </div>