Parts:Engineering
module 1.basic
C1
The construction of recombinant plasmids was carried out sequentially, including recombinant plasmid pET46a-ArsR-sfGFP-V0, recombinant plasmid pET-46a-PlacV-ArsR-ParsOC2-sfGFP-V1, and recombinant plasmid pET46a-ParsOC2-ArsR-sfGFP-V2.
According to the parameters commonly set in the article, the performance of the biosensor was tested, and its fluorescence intensity and OD value were measured separately using a microplate reader.
The results showed that in the arsenic-free condition, green fluorescent protein (GFP) exhibited a relatively high background expression. Meanwhile, combined with the literature reports, it is known that the expression level of ArsR protein driven by the constitutive promoter affects the sensitivity of the biosensor. Therefore, it was decided to optimize this constitutive promoter.
C2
The construction of recombinant plasmid pET46a-PlacV-ArsR-ParsOC2-sfGFP-V3 and recombinant plasmid pET-46a-PJ100-ArsR-ParsOC2-sfGFP-V4.
Construction of plasmids V3 and V4
The results showed that in the arsenic-free condition, the background expression level of GFP remained at a relatively high level without significant changes. By predicting the binding effect between the promoter and the repressor protein, and combining the results of the first round of cycles, it is inferred that this phenomenon may be due to the poor binding efficiency between the two, and this hypothesis still requires further experimental verification.
C3
To verify whether the binding efficiency between the repressor protein ArsR and the promoter is a key factor affecting the background signal intensity, we conducted multi-gradient induction control experiments using DH5α strains transfected with plasmids V3 and V4. The purpose of these experiments is to rule out interfering factors such as unreasonable concentration gradient range of inducers, inappropriate initial induction concentration, and excessively long or short induction time.
multi-gradient setup
To verify whether the binding efficiency between the repressor protein ArsR and the promoter is a key factor affecting the background signal intensity, we conducted multi-gradient induction control experiments using DH5α strains transfected with plasmids V3 and V4. The purpose of these experiments is to rule out interfering factors such as unreasonable concentration gradient range of inducers, inappropriate initial induction concentration, and excessively long or short induction time.
The performance of the biosensor was tested, and its fluorescence intensity and OD value were measured separately using a microplate reader.
module 2.Promoter engineering
C4
Design and use the native promoter from CML2, and attempt to test its binding effect with the ArsR protein.
Construction of plasmid pET-46a-PJ100-ArsR-ParsCML2-sfGFP-V4
It is necessary to construct a promoter library and conduct screening.
C5
D:构建启动子文库
B:
T:性能检测
L:
module 3.Self-assembling system
C6
Through logical analysis, leakage is inevitable in this genetic circuit; however, there exists a systematic leakage efficiency, defined as the ratio of "leakage brightness to total brightness". Therefore, we aim to design an AND-gate logic circuit to reduce the overall leakage efficiency, thereby achieving the goal of lowering background expression.
By reviewing relevant literature, we selected GFP self-assembly tags with proven performance, which were respectively constructed downstream of the promoter ParsOC2 in the circuit. After culturing the system for a specific period, induction was initiated.
Following a certain induction duration, a microplate reader was used to measure fluorescence intensity. The data were then collected, analyzed, and used to generate corresponding curves.
C7
Express and purify GFP1-10 and GFP11 separately in vitro, perform fusion experiments in vitro, and verify the analysis in C6.
Purify the 6His-GFP1-10 and 6His-GFP11 fusion proteins respectively using nickel column affinity chromatography, and then mix the two for reaction.
The target fragments of GFP1-10 and GFP11 were not successfully obtained through purification.
It is inferred that the purification failure of the target fragments may be due to two reasons: first, the molecular weight of the fragments is too small; second, the N-terminus or C-terminus of the fragments was not capped for protection, leading to degradation during the purification process.