Part:BBa_K4822007
pSB1C3-Phla-mCerulean (downstream Calprotectin Biosensor)
Introduction
The biomarker Calprotectin consists of protein subunits S100A8 and S100A9 that can bind to zinc ions (Zn2+). It is related to diseases like rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease.In order to construct an E. coli that can detect the biomarker Calprotectin, we adopted the Two-Component System(TCS) SaeRS from Staphylococcus aureus.
The SaeRS system of Staphylococcus aureus is composed of two proteins, SaeR and SaeS, which work together to detect environmental signals and regulate downstream gene expression. When the sensor histidine kinase SaeS phosphorylates the response regulator SaeR, the phosphorylated SaeR binds to a direct repeat sequence in specific downstream promoters, activating gene expressions. However, in the presence of Zn2+, the phosphorylation of SaeS is inhibited, preventing the phosphorylation of SaeR and consequently inhibiting downstream gene expression. There are studies that show when Calprotectin binds Zn2+, it could effectively prevent downstream gene expressions from being inhibited. This implies that when Calprotectin is present, it would increase downstream gene expression.
Usage and Biology
We apply the aforementioned mechanism to our experiment design. Phla is the promoter of the hla gene in Staphylococcus aureus. It is one of the promoters that is regulated by the two-component system SaeRS. The experiment concept we adopted is that under the presence of a higher concentration of Calprotectin, there would be fewer Zn2+ free ions. With fewer Zn2+ free ions, the expression of the gene for the fluorescent protein mCerulean would increase, producing a higher fluorescent intensity for us to detect. This way, we can infer the concentration of Calprotectin by observing the fluorescent density emitted by the E. coli.
Plasmid Engineering
We ligated the five genes, saeRS, Terminator, Origin+CmR, phla, and the gene for mCerulean, for our construct design using Gibson Assembly and transformed the engineered plasmid into E. coli. We adjusted the proportion of each part until we succeeded in cloning our genes into an assembled plasmid.
After we transformed the Gibson Assembly ligation product into E. coli, we cultured the transformed bacteria on solid media with antibiotic Chloramphenicol(Cm). In order to confirm our ligation result, we picked several colonies and amplified them in liquid LB + Cm media to perform plasmid extraction. From Figure below, we see that colony 16 had the desired complete plasmid.
We cultured bacteria from colony 16 in liquid media to double-confirm our result. From Figure below, we can be certain that we successfully constructed the pSB1C3-phla-mCerulean-p3-SaeRS plasmid and transformed it into E. coli.
The wells labeled “S” are the pSB1C3-phla-mCerulean-p3-SaeRS plasmid,
while wells labeled “C” are the pSB1C3-J364007_Promoter_RBS_T1_T2_with_mCerulean_(hGFP)_CDS plasmid.
The C groups serve as a comparative reference to ensure the functionality of the digestive enzymes.
SDS-PAGE
We extracted protein from the successfully transformed bacteria and performed SDS-PAGE. Due to our primer design, we only added one His tag after the SaeS protein in our plasmid construct. Since the genes for SaeR and SaeS are situated downstream of the same promoter and the two genes adjoin, confirming the expression of one protein is to ensure the expression of the gene saeRS. Therefore, we only performed protein purification and SDS-PAGE on the SaeS protein. From the Figure below, we can see that the bacteria expressed the saeRS gene and produced the protein.
Protein function
To investigate the sensitivity of our Sae plasmid device, we conducted fluorescent intensity and OD600 measurements to investigate the relationship between fluorescent density and Zn2+ concentration. We performed the Zn2+ test twice, eliminating data with OD600 measurements below 0.2 to mitigate significant errors caused by the limited bacterial numbers. We assume that the growth problem in high Zn2+ concentration groups is due to the high osmotic pressure. Within the range of 1 to 1.4mM Zn2+ concentration, there is a notable correlation between fluorescent density and Zn2+ concentration. But the trend in fluorescent density is not distinct for Zn2+ concentrations below 1mM. We realized our device does not have the function we anticipated in low Zn2+ concentrations. Therefore, we plan to modify the promoter of the saeRS gene to improve the sensitivity of engineered bacteria. However, our experiment only measured fluorescent density in 10 different Zn2+ concentrations. We plan to conduct more thorough experiments to collect more comprehensive data to determine the sensitivity of our plasmid device.
Reference
[1] Liu, Q., Yeo, W. S., & Bae, T. (2016). The SaeRS Two-Component System of Staphylococcus aureus. Genes, 7(10), 81. https://doi.org/10.3390/genes7100081
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 94
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |