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).
Step 7:- Fluorescence was taken for three samples (LB media, 2mM, and 5mM).
Result: - No conclusive result, control is showing higher fluorescence (maybe autofluorescence).
Step 8:- Protein isolation from cultures: Materials: - Phosphate Buffer (20mM Sodium Phosphate, 50Mm Nacl), 8M Urea Method: - Cultures were pelleted, the supernatant was discarded. 200uL 8M urea was added to the pellet, the pellet was pipetted until it was dissolved. 2 mL Phosphate buffer was added to it (make up the volume to level up for the fluorescence cuvette). The next step done was to centrifuge it at 6500 rpm for 5 min. Now carefully pipette out the supernatant into a fresh MCT without disturbing the pellet
Step 9:- Fluorescence (0mM (ctrl), 2mM, and 5mM). Result: - No conclusive result, control (0mM uninduced) shows similar fluorescence as induced, and 5mM IPTG shows lower fluorescence than 2mM.
Step 10: -Since DH5 alpha cells are used as cloning strain we thought to repeat all steps in Expression strain BL21 (DE3) pLysS. It also contains a plasmid, pLysS, which carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target genes under the control of the T7 promoter but does not interfere with the level of expression achieved following induction by IPTG.
Step 11:- 1 ul Plasmid isolated in Step4 were transformed into strain BL21 (DE3) pLysS. Result:-
Step 12:- Primary culture of 5 ml using 1 isolated colony, and let it grow at 37 degrees C for 12 hours.
Step 13:- 1 per cent of primary culture is used for secondary culture in 4 (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, 3mM, 5mM. After that cultures were induced at 37 degrees Celsius for 12 hours.
Step 14:- Protein was isolated using Step 8
Step 15:- Fluorescence was measured (0mM (ctrl), 3mM, and 5mM).
Result: - Correct pattern observed, control (0mM uninduced) shows lower fluorescence in comparison to the induced culture, and 5mM IPTG shows the highest fluorescence. But the difference was not much.
Step 16:- Currently we are working on Expression strain BL21 (DE3) standard and BL21 Lemo21 (DE3) for clear results.
Step17:- 1 ul Plasmid isolated in Step4 were transformed in both strains.
Step 18:- Primary culture of 5 ml using 1 isolated colony, and let it grow at 37 degrees C for 12 hours.
Step 19:- 1 per cent of primary culture is used for 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, 5mM. After that cultures were induced at 37 degrees Celsius for 12 hours.
Step 20:- Protein was isolated using Step 8
Step 21:- Fluorescence was measured (0mM (ctrl), 2mM, and 5mM). Results: Step 18:- Primary culture of 5 ml using 1 isolated colony, and let it grow at 37 degree C for 12 hours.
(Note:- For all the work (in plates, primary and secondary culture) antibiotic used is chloramphenicol.)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contributions:
Group: iGEM_TAU 2022
Summary: We have generated an optimized version of this construct, utilizing the ESO software (developed by TAU representative group for the iGEM 2020 competition), by the means of evolutionary stability of it: improving its ability to preserve functionality over long periods of time by avoiding loss-of-function mutations, taking into account different aspects, such as kinetic and thermodynamical parameters and Codon-biases. A link to our parts improvement page: https://parts.igem.org/Part:BBa_K4219001.
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