Part:BBa_K4632014
EGFP
Description
Aequoria victoria green fluorescent protein(Provided by Prof. Yongyao Xie, yyxie@scau.edu.cn).
What have we done? (SCAU-China 2023)
We characterized its performance in E. coliTOP10. The results can be found in our composite part-Part:BBa_K4632024[1]
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]
Construction and Characterization
(1) Characterization of Reporter Gene eGFP
Single colonies of engineered bacteria were selected and inoculated into LB broth. After overnight growth, the OD600 was adjusted to 0.6. Ara at a final concentration of 0.02% was added, and the cultures were incubated for 6 hours. eGFP fluorescence intensity was measured using a microplate reader (excitation wavelength 488 nm, emission wavelength 506 nm).
Blank control group: sterile LB, control group: engineered bacteria without 20% ara added, experimental group: engineered bacteria with inducer added, 3 replicates per group. There was no significant difference in fluorescence intensity between the control group and the experimental group, indicating no expression of eGFP
(2) eGFP Protein Expression Experiment
Single colonies of engineered bacteria were selected and inoculated into LB medium with inducer for overnight incubation. Top10 subjected to the same procedure served as the control group. Bacterial pellets were collected, and SDS-PAGE experiments were conducted, revealing no expression of eGFP at the protein level.
'(3) q-RT-PCR Experiment'
q-RT-PCR experiments were conducted under various conditions, confirming no expression at the mRNA level.
The reporter genes could not be expressed. The characterization of reporter genes eGFP and RFP was performed simultaneously with the second verification system. Consistent results were obtained from SDS-PAGE experiments and q-RT-PCR experiments.
We hypothesize several possible reasons:
1. The selected reporter genes eGFP and RFP may not be suitable for expression in Top10.
2. Errors may have occurred in the design of the gene expression module.
3. Interactions between genes may have occurred.
See more detail in BBa_K4632024[2]
| None |