Summary:

We were able to perform experiments to characterize some of the parts in our project design. We performed an experiment to test the working of our ironQS system and measured its ability to first be controlled by the amount of iron in vitro by measuring fluorescence of GFP under the control of our new luxI + FUR box promoter (BBa_3031015). We further tested the system’s ability then to express the ORF81 antigen. We were also able to perform fluorescence experiments aimed at characterizing existing parts already in the registry that can sense and regulate the expression of genes in response to oxidative stress. Due to the unfortunate situation where our sponsor could not assist us with our experiments, we were unable to test the cell wall anchoring system or perform the assays for characterizing the signal peptides. These parts were planned to be characterized using experiments with fluorescent microscopy. These parts have been sequenced and are now in storage in our lab for future characterization after the jamboree.

We first performed transformation of an expression vector containing our genes of interest. The DNA was synethzued by Genscript China and we used expression vector pET30a(+). Cells were cultured in our lab and expression was achieved through regulation of the amount Fe (II) in the culture. We test the working of iron QS system by measuring the intensity of fluorescent using plate reader. To detect the production of ORF81, we applied SDS-page to separate proteins based on the molecular weight and used western blotting for qualitative analysis. Finally, we modified and tested the H2O2 promoter previously designed by Team NYMU-Taipei.

Protocols:

Iron QS system testing

Preparation:

Plasmid construction

The sequence of our target gene cluster was obtained from NCBI. Prior to synthesis onto plasmids by Genscript™ we edited the sequence to remove all illegal restrictions sites to ensure our biobricks would be RFC10 compatible.

Transformation of pET30a (+) vector:

Transformation of the synthesized expression vectors into BL21 (DE3) high efficiency competent cells were achieved through Heat Shock method. Protocol from NEB. Successful transformation was observed on LB agar plates containing Ampicillin antibiotic marker (working condition 50μl/ml). Positive control (BL21 cells plated on LB agar plates w/Ampicillin) showed cells were viable, while Negative control (BL21 cells w/o plasmid plated on LB agar plates containing Ampicillin) yielded no colonies. All plates were incubated for 16 hours overnight at 37 C.

Expression of Genes of interest.

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.

Chemical structure of 2,2'-Dipyridine (left) and its iron chelating properties (right)

Meseaurment

Expression assay of iron QS strains in vitro

For our primary lab, we replace antigen with reporter gene GFP for testing the system. Therefore, we design the circuits contains GFP gene which is used as a reporter of expression with an appropriate promoter. Ideally, we may detect significant relative fluorescence value difference bewteen sample B and sample A &C . 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 A, B, C was added into 96 well white polystyrene microplate and black polystyrene microplate, each with three samples. We measured the OD600 and Fluorescence by using plate reader. The data was recorded. After that, we calculate the average OD600 and Fluorescence for Sample A, B, and C. 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.

SDS-Page

A 15mL of BL21 ORF81 is grown overnight (18 hours), and another 15mL of ORF81 is grown for 4 hours, representing different cell density. 15mL of high-density ORF81 culture is divided into 3 tubes, each contains 5mL. 15mL of low-density culture is then repeated with this process. 1.5mL sample is taken from each tube and centrifuged in 8000rpm (5700g) for 1 minute. 1mL solution A, 1μL solution B, 10uL solution C are added to each sample per 20 mg wet weight. Since solution A is stored in -20 Celsius degree, it is possible for appearance of precipitation. 30℃ water bath could be used to completely dissolve solution A. Use of vortex mixer ensures the resuspension of culture. Then all samples are put on rocker for 10 minutes in room temperature. Free thawling (FT) method using liquid nitrogen then frees protein completely. After cool down, in 4 Celsius degrees, 6 samples are centrifuged with 12000rpm for 5 minutes. The supernatant is removed to another tube for later experiment.

10× SDS-PAGE Sample Loading Buffer is dissolved in room temperature and added into sample with ratio of 1:9. The mixture is then put in boiled water bath for 4 minutes in order to completely denature protein. After the samples cool down in room temperature, 100μL of each is loaded into the gel. Stop electrophoresis when the color reaches the bottom line. The gel is taken out and put in dye solution. After two days, photo the gel.

Western Blotting

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 Celsius degrees. 10ml culture was added to each of three 15ml tubes. Sample A contains culture (with plasmid) with 200 μM DP (2,2'-Bipyridine). Sample B contains only the culture (with plasmid).

After induction with DP for 4 hours, 1.5 ml of each cell culture broth was transferred to three 1.5 ml sterile centrifuge tubes and total 6 tubes are centrifuged in 8000rpm (5700g) for 1 minute. 1mL solution A, 1μL solution B, 10μL solution C are added to each sample per 20 mg wet weight. Since solution A is stored in -20 Celsius degree, it is possible for appearance of precipitation. 30℃water bath could be used to completely dissolve solution A. Use of vortex mixer ensures the resuspension of culture. Then all samples are put on rocker for 10 minutes in room temperature. Free thawling (FT) method using liquid nitrogen then frees protein completely. After cool down, in 4 Celsius degrees, 6 samples are centrifuged with 12000rpm for 5 minutes. The supernatant is removed to another tube for later experiment.

6× SDS-PAGE Sample Loading Buffer is dissolved in room temperature and added into sample with ratio of 1:5. The mixture is then put in boiled water bath for 4 minutes in order to completely denature protein. After the samples cool down in room temperature, 100μL of each is loaded into the gel. The voltage is constant 80V and changed to 120V after the sample reaches the lower part of gel. Stop electrophoresis when the color reaches the bottom line. The gel is taken out.

We make Western Transfer Buffer by mixing 11.52g glycine, 2.424g trace, 200mL methanol. Double distilled water is added to 1L. The gel part with sample is cut out. A same-sized nitrocellulose filter membrane is cut and two larger filter papers are prepared. All of these materials and gel are soaked in Transfer Buffer. A “sandwich” is made by stacking Sponge, Filter paper, NC membrane, Gel, Filter pater, Sponge in blotter unit. In ice, electrophoresis is conducted for the blotter unit with 60V for one hour.

Then, for membrane blocking, the membrane is blocked in blocking solution (5mL non-fat, dry 5% milk in TBST buffer) for 1 hour in 4 Celsius degrees. The membrane is washed in TBST for 1 minute after incubation with blocking solution. The primary antibody (His) solution is diluted with 5mL TBST in 1:200. The membrane is incubated in primary antibody solution overnight at 4°C. Incubation of membrane in antibody solution with rocking motion facilitates even binding. After incubation, the primary antibody solution is removed. The blocking solution is diluted to 3% and added to membrane for 1 hour. The membrane is washed with TBST in three washes of 4 minutes each. Then secondary antibody solution is diluted with TBST in 1:1000. The membrane is incubated in secondary antibody solution for 1 hour at room temperature with agitation. After incubation, the secondary antibody solution is removed. The membrane is washed with TBST in three washes of 4 minutes each. The final picture is taken with X-Ray.

H2O2 sesnor testing

Introduction:

We designed a potential improvement to the OxyR transcription factor protein, which when activated by a reactive oxygen species (ROS) undergoes a conformation change which activates it to positively active certain ROS sensitive promoter sequences. This transcription factor is thus then said to be a regulator of certain promoters. We measured the fluorescence of GFP under the influence of the TrxC promoter which is activated by OxyR in the presence of H2O2. We also incorporated our attempt at an improved design into the sequence and took the same measurements. Please see our model page for extensive details of how we made changes to this part for potential improvement.

Day1:

- Plasmid Construction

We asked Genscript™ to help us construct two plasmids containing different transcription factor OxyR under the influence of a constitutive promoter (BBa_J23102) along with a reporter gene GFP (BBa_E0840) under the influence of the ROS induced promoter TrxCp (BBa_K1104201). This construct would one express GFP in the presence of a ROS like H2O2 as OxyR would then be activated and positively influence the TrxC promoter. The OxyR sequence was added to the registry by NYMU-Taipei's 2013 iGEM team (Part:BBa_K1104200) while the new OxyR sequence is our modified version (Part:BBa_K3031018).

- Transformation:

Transformation of the synthesized expression vectors into BL21 high efficiency competent cells was achieved through Heat Shock method. Protocol from NEB. Successful transformation was observed on LB agar plates containing Ampicillin antibiotic marker (working condition 50μl/ml). Sample A is the gene circuit conaining the original OxyR sequence (BBa_) and Sample B is the circuitcontaining our new OxyR sequence part (BBa_). All plates contained both samples were incubated for 16 hours overnight at 37 °C.

- Overnight Culture:

We selected single colonies from both samples A and B and cultured overnight in four 50 ml LB broth containing Ampicillinin a 1000:1 ratio at 28 °C

Day2:

Pre-Lab Preparation

We divided four 50ml test tubes of Sample A and B culture 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:

Tube	Concentration of H2O2added	Final Contents
A1	5mM	5ml A+ 5ml LB+ 5.10μl H2O2
A2	2.5mM	5ml A+ 5ml LB+ 2.25μl H2O2
A3	1mM	5ml A+ 5ml LB+ 1.02μl H2O2
A4	0.1mM	5ml A+ 5ml LB+ 0.10μl H2O2
A5	0.01mM	5ml A+ 5ml LB+ 0.01μl H2O2
A6	0mM (Control)	5ml A+ 5ml LB
B1	5mM	5ml B+ 5ml LB+ 5.10μl H2O2
B2	2.5mM	5ml B+ 5ml LB+ 2.25μl H2O2
B3	1mM	5ml B+ 5ml LB+ 1.02μl H2O2
B4	0.1mM	5ml B+ 5ml LB+ 0.10μl H2O2
B5	0.01mM	5ml B+ 5ml LB+ 0.01μl H2O2
B6	0mM (Control)	5ml B+ 5ml LB

Measurement:

After 2 hours of incubation, we extracted 1.4 ml culture from each of tubes and placed in twelve 1.5ml sterile centrifuge tube and centrifuged at 4000rpm for 4 minutes. After removing the supernatant, we washed the cell with PBS buffer. Then, 200 μM culture of A1, A2, A3, A4, A5, A6, B1, B2, B3, B4, B5, B6 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 calculated the average OD600 and Fluorescence for all samples. For each of samples, we divided the relative fluorescence value (RFV) by the average OD600. This quantitative test was to add new data for the old part designed by Team NYMU-Taipei and make comparison with our improved part with the modified OxyR transcription factor.