Part:BBa_K2913018

Anti-ECF20(optimized by E.coli)

A protein that inhibits the initiation of transcription of ecf20

result

Fig. 1(a). Green fluorescence intensity of produced GFP driven by the Pecf20 promoter. Plasmids pYBIa-ECf20 & pRB1s-Pecf20-EGFP were transformed in to E. coli Nissle 1917, and the bacteria were cultured at 37 °C in medium containing 0.5 mM Ara and 0.4 g/l glucose. OD600nm and green fluorescent signal were measured every 10 min using an automatic microplate reader. (b). Green fluorescence intensity of produced GFP to determine the antagonism of Anti-ECF20 to the Pecf20 promoter. Plasmids pYB1a-ECf20, pRB1s-Pecf20-EGFP and pSB1c-Plac-Anti ECF20 were individually transformed into E. coli Nissle 1917, and The bacteria were cultured in LB medium containing 0.5 mM Ara and 0.4 g/l glucose. IPTG was added to different final concentrations of 0, 1, 5, 10 and 20 mM. The bacteria were then cultured at 37℃ with the measurement of OD600nm and green fluorescent signal every 10 min using an automatic microplate reader.

In Fig. 1(a), when pYB1a-p-BAD-EGFP (triangle), pYB1a-ECF20 & pRB1s-Pecf20-EGFP (square) was respectively transformed into E. coli Nissle 1917, green fluorescence intensity increased in a time dependent manner. When pRB1s-Pecf20-EGFP (circle) was transformed into the bacteria, green fluorescence intensity did not show obvious increase with time. We confirmed that ECF20 is a necessary factor for the activation of the Pecf20 promoter. In Fig. 1(b), the addition of IPTG led to dramatic difference of detected green florescence signal. Without IPTG, we detected the continuously increased green fluorescence signal with the time. On the other hand, with the IPTG addition, the fluorescence signal remained at the baseline level. The data confirmed that Anti-ECF20 could suppress the activity of Pecf20 promoter. Overall, our experiments indicated that ECF20 is necessary for the activation of the Pecf20 promoter, while Anti-ECF20 can repress the Pecf20 promoter through binding to ECF20 and suppressing its function.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 295
  
• 1000
  COMPATIBLE WITH RFC[1000]