Team:SUIS Shanghai/Demonstrate
PARTS
HUMAN PRACTICES
Demonstrate
Overview
- We believe the experiments we have done with some of our composite parts are good enough for this medal criteria. The first goal of our experiments was to only express downstream genes in an iron starved environment. This environment was chosen to mimic the guts of fish so our system would be able to express an antigen capable of eliciting an immune response in the fish mucosal tissue. We first used part BBa_K3031016 which is our constructed system (where expression of downstream genes depends on two factors: 1. High cell density caused by the normal LuxR/LuxI system and 2. Low ferric iron in the surrounding environment thanks to our new basic part BBaK303105 which has a FUR box sequence inserted into the LuxI promoter. This system means at high cell densities, the LuxR/LuxI system does not work as expected due to repression of LuxI and the subsequent absence of AHL (inducer of the Lux promoter). This first composite part used GFP as a reporter gene to test whether E.coli BL21(DE3) cells would express GFP only at low iron concentrations (i.e. GFP would be suppressed in media containing iron).
After gaining positive results from this test we also then tested the new part BBa_K3031017 which is the exact same system as above, with the exception that the reporter GFP gene is replaced by a coding region for a membrane protein of the Cyprinid herpes virus-3 (the pathogen linked to Koi herpes virus disease). To achieve low iron environments for both experiments we cultured cells with DP (2,2'-Dipyridine), which is a string iron chelator. Both results described below shows that our system works in conditions of low iron which mimic the gut of fish and therefore are promising system to be used in live engineered bacteria vector system for the expression of recombinant antigens.
Experiment
To test the effectiveness of our new part luxI promoter with FUR - we needed to expose cells containing transformed plasmid into both iron rich and iron starved environments. Single colonies were inoculated in 50 ml LB broth containing Ampicillin in a 1000:1 ratio and 40 μM FeSO4 in Falcon tubes and cultured at 37 C until OD600 = 0.5. 10ml culture was added to each of three 15ml tubes. Sample A contains blank cell (without plasmid) culture. Sample B contains culture (with plasmid) with 200 μM DP (2,2'-Dipyridine). The function of the 2,2'-Dipyridine is to remove iron in the cellular environment and thus mimic the low iron environment of the gut. Sample C contains only the culture (with plasmid) without any 2,2'-Dipyridine.
After induction with DP for 4 hours, 1 ml of each cell culture broth was transferred to two 1.5 ml sterile centrifuge tubes and centrifuged at 4000rpm for 4 minutes. After removing the supernatant, we wash the cell with PBS buffer. Then, 100 μM culture was added into 96 well white polystyrene microplate and black polystyrene microplate, each with three samples. We measured the OD600 and Fluorescence (Excitation: 485nm/ Emission: 528nm) by using plate reader. The data was recorded. After that, we calculate the average OD600 and Fluorescence for each sample. For each of samples, we divided the relative fluorescence value (RFV) by the average OD600. This quantitative test was used to determine Fur and luxI/luxR-controlled protein expression under iron deprivation in E. coli.
- Sample A = Blank (E.coliBL21(DE3) cells with no plasmid)
- Sample B = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media PLUS iron chelator 2,2'-Dipyridine
- Sample C = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media only.
| Blank | Iron QS+DP | Iron QS | |
|---|---|---|---|
| RFV(AVG) | 952237 | 397057 | 554270 |
| OD(AVG) | 0.604 | 0.119 | 0.343 |
| RFV/OD600 | 1576551.325 | 3345985.955 | 1614379.612 |
Western Blotting
Figure 3: Western blotting result of Iron-QS system expressing ORF 81. Lane 1, 2, and 3 are three repititions of sample A, and lane 4, 5, 6 are three repititions of sample B. As the result of western blotting indicated, three lanes of sample A share the same polypeptite band, so do three lanes of sample B. This suggests a difference in protein expression between sample A and B, which is a result of induction and repression of our system. Iron QS in sample A is ideally expressed as the iron chelator-DP-reduce the ferric iron concentration in the medium. The sytem in sample B is repressed by iron-bound holo FUR. However, three possible bands for protein of interest corresponds to 43 kDa molecular on the ladder. Although there's a difference between the result of western blotting and our ideal protein size (29 kDa), this might be caused by post translational modification of protein. Possible chemical modification, such as glycosylation, methylation, and phosphorylation, may contribute to the variance of protein size. Most membrane-bound proteins expressed in the endoplasmic reticulum are glycosylated, which entail covalent addition of sugar moieties to specific amino acids, to some extent [1]. Because the oligosaccharides could be very large, it's possible the bands are results of glycosylation of our protein of interest.
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