Part:BBa_J04421

ECFP Coding Device with Promoter, RBS, Coding Sequence, and Terminator

IPTG Inducible Promoter, RBS, ECFP with LVA tag, Terminator

Usage and Biology

This part will be made using standard digestion and ligation techniques described in the "Registry of Biological Parts" website.

IISER Bhopal 2021

Step 1:- Extracting DNA out from the kit plate (15P).

Step 2:- Extracted DNA was transformed into DH5 Alpha cells.

Step 3:- 10 ml of primary culture was grown using 1 isolated colony, and it was grown at 37 degrees C for 12 hours.

Step 4:- Primary culture is divided into two parts, one is used for plasmid isolation and the other is used for secondary culture.

Step 5:- 1 per cent of primary culture was used to set the secondary culture in 3 (15 ml) falcons in 5 ml media and after approximately 2.5 hours when OD reached around 0.6, IPTG was added at different concentrations, 0mM, 2mM, and 5mM. After that cultures were induced at 37 degrees Celsius for 12 hours.

Step 6:- 2mM Culture was pelleted, the supernatant was discarded. 20 uL 8M urea was added to the pellet, the pellet was pipetted until it was dissolved. 5 uL protein loading dye (containing Beta mercaptan) was added to it. The sample was heated for 10 minutes at 80 degrees. Samples were run on SDS gel.

Results: The following image was obtained in the gel doc. A dark prominent band was obtained around 25 KdA. The size of ECFP was 25KdA (literature survey).

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]