Overview

    This year, we have done many quantitative measurements to evaluate our work, to make our experiment more credible, to characterize our parts and to verify the feasibility of design. Based on the principle that protiens can reduce copper ions to Cu+, which can combine with BCA to form a purple-colored reaction product, we used microplate reader to detect the concentration of proteins of loading sample in Western Blot. In addition, to characterize the enzymatic activity of our SAMe synthetase, we use enzyme-linked immunosorbent assay (ELISA) to measure SAMe concentration at different time. In safety switch module, a microplate reader was used to detect green fluorescence, whose intensity reflected function or expressive level of Temp and Chem. And in order to verify the lysis function of PBSX holin, we quantified OD 600nm of bacteria concentration at each time point. Meanwhile, the improvement of the RBS is also based on this quantitative data of fluorescent intensity of reporter GFP. To test the hardware Portable Dual Port Filter, we used spectrophotometer to measure OD600 of the bacterial solution in both treatment and control groups, so as to assess the feasibility of the device.These quantitative data make us clear about the improvement direction to deepen the project and enable reuse building on the reported devices, systems, and protocols.

Standardization of Protein Concentration

Purpose

    In our project we used BCA quantification method to quantify the loading sample of our protein solutions before we performed Western Blot to ensure the concentration is proper (30-50ng) and the total protein mass remains the same in different wells so that we can compare the brightness strength of target bands to compare the concentration of the protein of interest. In this case, we can figure out whether the expression level of protein of interest can increase along with induction time, induction concentration, incubation temperature and other variables. With such quality control, we can explore the best production condition or induction efficiency.

Principle

    The BCA Protein Assay Kit is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu2+ to Cu+ by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) using a unique reagent containing bicinchoninic acid. The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562nm that is nearly linear with increasing protein concentrations over a broad working range (5–200 µg/mL).

Result

    We used the protocol in Protocol to draw a standard curve and measure the concentration of the protein of interest. The standard curve of opSam2 WB is showed in Figure 1.2A (Right), and the raw data of microplate reads are showed in Figure 1.2A (Left). The standard curve of opPet8p WB is showed in Figure 1.2B (Right), and the raw data of microplate reads are showed in Figure 1.2B (Left). After the BCA quantification, we performed dilution to standardize the total protein concentration before loading.

Figure 1.1 Principal Graph of BCA quantification

Figure 1.2 BCA Quantification and SAMe quantification Results.

Measurement of SAMe synthetase opSam2 using ELISA

Purpose

     In our project, we expressed an exogenous SAMe synthatase opSam2 to generate a natural anti-depressant SAMe. To characterize its enzymatic acitivity, we use SAMe quantification ELISA kit to measure the SAMe production in a certain condition.

Principle

    SAMe quantification ELISA kit, this assay employs the competitive inhibition enzyme immunoassay technique, and there is an inverse correlation between SAMe concentration in the sample and the assay signal intensity. Using microplate reader, we measured the signal intensity at 450nm of our induced engineered bacteria with opSam2. If proteins do work as we hope, we could find the signal declines as the induction time increases which means the SAMe concentration increases.

Figure 1.3 Principal graph of SAMe Quantification through ELISA.

Procedure

    First, we conducted our measurement on E. coli and B. subtilis. In pilot experimnet, we tried to use different concentration of IPTG to induce the expression of opSam2 and RIPA to lyse the bacteria so as to explode the produced SAMe. Through several rounds of pilot experiment, we chose 0h, 0.5h, 1.0h and 1.5h as induction time gradient. Along with the measurement of the samples, we built a standard curve to indicate the SAMe concentration of our samples clearly and to prove that our operation works as the manual said (Fig 1.4A).

Result and Analysis

    The formal experiment was conducted in B. subtilis and the concentration was calculated according to the standard curve (Fig 1.4A right table). We firstly analyzed the absolute concentration change of SAMe (Fig 1.4C). The concentrations in both Empty Vector (EV) group and pHT-Sam2 group increased along with increased induction time, but obviously, concentration of pHT-Sam2 group increased more sharply than EV group. So we think that this portion of increased concentration is due to the increased bacterial mass. So to exclude the effect of increased bacterial mass, we used EV group to standardize, and draw a graph dedicating the relative concentration change of SAMe in pHT-Sam2 group (Fig 1.4C). We can find out that opSam2 works normally since after induction, the relative concentration increased from Ratio = 1 to Ratio = about 2.5.
    In the latter induction phase, we found that the relative concentration of SAMe tends to be static. We suppose that the utilization of SAMe by bacteria could be upregulated as the source increased, which is benificial for bacterial proliferation since SAMe is an important one-carbon unit.

Figure 1.4 SAMe quantificatino and Normalization Result

Safety Switch Module

Purpose

    For biosafety consideration, we aimed to test the function of two composite parts which work as chemical and temperature switches respectively. To be more specific, we tested the fluorescence intensity of these composite part in both E. coli and B. subtilis. We also tested the strength of the cleavage of bacteria when PBSX holin expresses.

Principle

    With the induction of autoinducers, the chemical-switch will be activated. For chemical-induced switch, we treated engineered bacteria with CinI, a molecule for quorum sensing, to induce expression of Chem which can start downstream holin, a bacterial toxin. For temperature-controlled switch, in the environment below 36 degrees Celsius, it will work to also activate the expression of downstream holin.
    To determine whether they can work smoothly, we at first replaced holin with EGFP, intending to qualitatively verify by the production of fluorescence. Fluorescence microscopy was the method we chose. EGFP expressed can be excited by a single wavelength of light to produce green fluorescence, which can be captured by our eyes after passing through the filter. In addition, microplate-based measurements detect light signals from a sample.
    The signal is usually measured and converted by a photomultiplier tube (PMT) before being transported to the microplate reader. The output data can reflect the sample’s fluorescence intensity. Similar to the principle of a fluorescence microscope, the exciting light and emission light are selected by specific monochromators, which can monochromatize lights.
    To verify holin’s function, we overexpressed it by transforming pET-Holin into E. coli BL21 and utilized spectrophotometer, which outputs absorbance of solution at the wavelength of 600nm, to quantify the thallus concentration in the liquid.

Procedure

    For chemical-controlled switch, we used 1mM IPTG to induce BL21 to produce CinI and supernatant containing CinI was put onto thallus of E. coli and B. subtilis to activate Chem and EGFP. We set E. coli and B. subtilis without Chem-EGFP as control. Furthermore, we used microplate reader to quantify fluorescence intensity in B. subtilis.

    For temperature-controlled switch, after transforming vector into E. coli DH5α, we incubated engineered E. coli overnight and then equally divide bacteria into two EP tubes, cultured respectively at 25 and 37 degrees Celsius for the same certain time. The detection method was to use a fluorescence microscope to irradiate the bacteria liquid with blue light. Furthermore, we used a microplate reader to quantify fluorescence intensity in B. subtilis under 16, 25, 37 and 42 degrees Celsius.

     To verify holin’s function, we followed the procedures shown below.

Result

Chemical-induced switch

    With the fluorescence microscope, unfortunately, there was no green fluorescence.

Fig. 2.1: Fluorescence in E. coli with chemical switch

    Furthermore, we used microplate reader to quantify fluorescence intensity in B. subtilis.

Fig. 2.2: Measurement results of relative fluorescence intensity

    Relative to control group, values of chemical-induced + inducer increased to some extent, but the margin wasn’t obvious enough.

Temperature-controlled switch

     Our protocols and observation are shown below:

Fig. 2.3: Fluorescence in E. coli with temperature switch

    The short rod-shaped E. coli cultured at 25 degrees Celsius fluoresces green, while the bacteria cultured at 37 degree Celsius do not fluoresce. This indicates that Temp induced by low temperature can initiate the expression of downstream genes, while Temp does not work at high temperature. The results are in line with our expectations.

    We further wanted to test it in B. subtilis and quantitively measure the expression of fluorescence. Groups set are shown below:

Fig.2.4: Measurement results of relative fluorescence intensity

    Chart shows data after the second hour’s incubation. Values at 37 degrees are benchmarks. There was no significant increase in the data of the first two groups relative to the latter two groups, which means EGFP in engineered B. subtilis didn’t express.

holin

    The values can represent efficiency of holin.

Fig. 2.5: Measurement results of OD 600

    It can be seen that the bacterial concentration of the group induced by IPTG was significantly reduced compared with that of the control group, but there was no significant difference between different induced concentrations. The cleavage efficiency increased to some extent with the extension of culture time. The results showed that holin could work in E. coli.

Hardware

Purpose

    We planned to test our Portable Dual Port Filter for bacteria synchronization and separation by assessing its elution efficiency. To achieve that, we measured OD600 of the bacterial solution before and after passing through the filter membrane within the hardware, which represents the bacterial concentration.

Principle

    The spectrophotometer can generate light source of a specific wavelength. Part of the light is absorbed when passing through the test sample, and the absorption value of the sample is calculated, which is proportional to the concentration of the sample. At 600nm, the spectrophotometer is sensitive to turbidity and there is a linear relationship between the density of bacteria in the liquid and the optical density (OD) at this wavelength, so it can be used for quantification.

Procedure

    We conducted experiments according to procedures shown below (Fig 3.1). In the process, we centrifuged the bacteria solution before and after passing through the filter membrane within the hardware (Control group and Treatment group, respectively.) and resuspended the pellets in an equal amount of LB medium, incubating for 2h at 37℃. After incubation, we measured OD600 of the two groups using spectrophotometer, with LB medium for zero setting.

Figure 3.1 The procedure for hardware test.

Results

    We successfully finished four rounds of experiment and measurement, and calculated the elution efficiency based on our data. We firstly directly eluted the bacteria and then used the pellet to do the following measurement. However, we found that the elution efficiency was very low, as the adhesion of filtration was strong (Fig 3.2). So we improved the elution efficiency by rinsing the filter membrane into LB for elution. We surprisingly found that the elution efficiency increased a lot. The results are shown below (Table. 1 and 2).

Table. 1 Data before improvement.

Table. 2 Data after improvement.

Figure 3.2 Comparison of elution efficiency between [Direct Elution] and [Direct Elution + Rinse] Group.

    We kept the culture condition and operation the same and we supposed that OD600 could represent the bacterial concentration in a certain way for this experiment. So we here used OD600 directly to estimate the density of bacteria. The ratio of Filtered/ Control is considered to represent the elution efficiency. After the improvement from DE to (DE + Rinse), we could find that the elution efficiency probably increased for about 6 times. By analyzing these data, we have made plans for further improvement to the elution efficiency and convenience of the device in the future.