Difference between revisions of "Part:BBa K4645016"

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===Usage and Biology===
 
===Usage and Biology===
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<p>We transformed this biobrick into E. coli to regulate expression of genes. With the growing of bacteria, DPO accumulates. While the concentration of DPO reached the threshold value, the expression of NeuA, NeuB and TetR would be activated. Then TetR shut down the expression of amcyan in downstream.</p>
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<p>We transformed this biobrick into E. coli to regulate expression of genes. With the growing of bacteria, DPO accumulates. While the concentration of DPO reached the threshold value, the expression of NeuA, NeuB and TetR would be activated. Then TetR shut down the expression of AmCyan in downstream.</p>
 
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<center><img src=" https://static.igem.wiki/teams/4645/wiki/quorum-sensing/vqma-usage-and.png" style="width:75%; "></center>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/quorum-sensing/v-tus.png" style="width:65%; "></center>
 
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<center><b>Figure 1. The circuit we assembled. </b></center>
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<center><b>Figure 1. The composite part we assembled. </b></center>
 
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===Characterization===
 
===Characterization===
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<p>Blank BL21, BL21 transformed with this biobrick, were cultured in microplate reader 37°C, 220 rpm for 10 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.</p>
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<p>To test whether this biobrick can work as expected, we built a circuit that vqmA is under the control of lac operator, then transformed into BL21.
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We cultured blank BL21 (which had never been transformed any plasmid) and BL21 contain this plasmid till the OD600 of bacteria medium reached 0.6. Then BL21 with the plasmid were induced with 0mM IPTG、1mM IPTG. Whereafter, these 3 groups of bacteria were cultured in microplate reader 37°C, 220 rpm for 3.5 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.
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<center><img src="https://static.igem.wiki/teams/4645/wiki/quorum-sensing/v-tresult.png" style="width:50%; "></center>
 
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<center><b>Figure 2. Fluorescence intensity / OD600 in BL21 and <i>luxR-tetR</i> </b></center>
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<center><b>Figure 2. The changes of fluorescence intensity / OD600 in blank BL21, 0mM IPTG insulted, 1mM IPTG insulted over time.  </b></center>
 
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<p>The result showed that in the early growth stage, transformed bacteria expressed amcyan fluorescent protein continually. When the group density reached the threshold value, the expression of amcyan fluorescent protein began to be blocked up.</p>
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<p>Unluckily, induced bacteria showed stronger fluorescence intensity than control one, which is contrary to the expected result. We speculate that it might lead by the malfunction of qtip promoter.</p>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 12:56, 12 October 2023


J23100-B0030-vqmA-B0017-Pqtip-tetR-B0015-PTet-amcyan-B0017

Verification of Not-Gate.


Usage and Biology

We transformed this biobrick into E. coli to regulate expression of genes. With the growing of bacteria, DPO accumulates. While the concentration of DPO reached the threshold value, the expression of NeuA, NeuB and TetR would be activated. Then TetR shut down the expression of AmCyan in downstream.

What？


Figure 1. The composite part we assembled.

Characterization

To test whether this biobrick can work as expected, we built a circuit that vqmA is under the control of lac operator, then transformed into BL21. We cultured blank BL21 (which had never been transformed any plasmid) and BL21 contain this plasmid till the OD600 of bacteria medium reached 0.6. Then BL21 with the plasmid were induced with 0mM IPTG、1mM IPTG. Whereafter, these 3 groups of bacteria were cultured in microplate reader 37°C, 220 rpm for 3.5 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.

What？


Figure 2. The changes of fluorescence intensity / OD600 in blank BL21, 0mM IPTG insulted, 1mM IPTG insulted over time.

Unluckily, induced bacteria showed stronger fluorescence intensity than control one, which is contrary to the expected result. We speculate that it might lead by the malfunction of qtip promoter.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 599
    Illegal AgeI site found at 2547
  • 1000
    COMPATIBLE WITH RFC[1000]