Part:BBa_K4645016

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.

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.

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