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
Western blotting result of Iron-QS system expressing ORF 81. Lane 1, 2, and 3 are three repetitions 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. Because the oligosaccharides could be very large, it's possible the bands are results of glycosylation of our protein of interest.
Demonstration of H2O2 inducible gene circuit
We also demonstrated that two new composite parts containing a constitutive promoter expressing the transcription factor OxyR which when activated will positively regulate the TrxC promoter. In our system we tested the ability of OxyR to regulate this promoter by inserting a GFP generator downstream od TrxC promoter and performed an assay with different levels of H2O2. Our parts we submitted and demonstrated to work are BBa_3031020 (containing the original OxyR sequence) and BBa_3031019 containing a mutated version we made this year based on structural information gained about reactive cysteine environments. This protein reacts with H2O2 resulting in the oxidation of a reactive cysteine (Cys-199) formation of a intermolecular disulfide bond with neighboring cyctein-208 and thus a conformation change in the OxyR protein occurs. This change is shape activates the OxyR protein which then can bind to certain promoters with binding site regions to regulate the expression of downstream genes.
Experiment
We sent our new sequence to Genscript China to be sequenced, along with the original OxyR sequence. The circuit we used to measure any improvement in our cells is found below and consists of a constitutive promoter which always produced OxyR transcription factor protein plus a TrxC promoter (part BBa_K1104201) which contains two binding sites for OxyR and is activated for expression of upstream genes once OxyR reacts with ROS and forms the intermolecular disulfide bond. Finally to be able to measure the effectiveness of this system to be induced by ROS, a reporter gene GFP is added and is under control of the inducible promoter TrxCp (part BBa_K1104201). This composite part, although was theoretically described by NYMU iGEM 2013, was constructed and out into the registry as a new composite part (BBa_K3031020) by us (SUIS-Shanghai iGEM 2019). We provide characterization of the composite part below which also provides useful descriptions for the parts BBa_K1104200 (OxyR coding sequence) and BBa_K1104201 (TrxC promoter)
The OxyR gene is expressed by a constitutive promoter while the inducible promoter TrxC will only express downstream genes (GFP in this case) when it is activated by oxidized form of OxyR, by virture of binding to sites (maked in pink here). When H2O2 levels are high enough the OxyR is activated and GFP is produced. Both constructs were sequenced onto a expression vector pET301(+) and transformed the plasmid into Bl21(DE3) cells. To induce the TrxC promoter we divided four 50ml test tubes of OxyR and OxyR_Mutated into twelve 15ml test tubes, each contained 5ml culture broth and 5ml LB with OD600 0.4. H2O2 was added to 10 of the tubes in the following concentrations:
Where:
- Sample A = E.coli BL21(DE3) cells containing expression plasmid pET30a(+) containing: Composite part with original gene sequence of OxyR (this part) under influence of constitutive promoter and GFP under the influence of inducible promoter TrxCp
- Sample B = E.coli BL21(DE3) cells containing expression plasmid pET30a(+) containing: Composite part with mutated gene sequence of OxyR (BBa_K3031018) under influence of constitutive promoter and GFP under the influence of inducible promoter TrxCp
- Blank = E.coli BL21(DE3) cells containing expression plasmid pET30a(+) containing no engineered plasmid.
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