Team:NCTU Formosa/Measurement

To compete the prize of best measurement, this year, we measured the function of biobrick BBa_I6060, a kind of yellow fluorescent protein, and the quorum sensing system as our measurement. For the yellow fluorescent protein, first, we resuspended the DNA from the distribution kit and then transformed it into E. coli BL21. Since there is LacI gene inside the bacteria, the yellow fluorescence protein will be restricted due to the Lac promoter, normally. If we use IPTG to induce the bacteria, the LacI promoter will be opened and produce the yellow fluorescence protein. After transformation, we will measure the optical density and fluorescence intensity by the ELISA reader - CLARIOstar®. For the quorum sensing system, we cloned the quorum sensing gene into the E. coli and did the functional test.

I. Biobrick BBa_I6060 characterization

1. Experiment Design

We plan to separate our experiment into two parts: first is colony PCR, which are used to see if we produce our target gene or not. The second part is the optical density and fluorescence intensity, and they are used to confirm the function of the yellow fluorescence protein.

A. Colony PCR

In this part, we confirmed the nucleotide sequence size and protein size by using colony PCR. First, we get the biobrick BBa_I6060 from the distribution kit and then transform it to E. coli DH5α. Then we ran both experiments simultaneously. The colony PCR protocol can be seen on our pWe confirm the sequence size and protein size by using colony PCR. First, we get the biobrick BBa_I6060 from the distribution kit and then transform it into E. coli BL21, then we do both experiments simultaneously. The protocols of PCR can be seen in the protocol page of our wiki.

B. Optical Density and Fluorescence

Confirming the size of the sequence and the protein size is not enough for us to make sure the protein works fine in E. coli. Therefore, we measure the optical density value and fluorescence intensity to make sure the functionality of our protein and the amount of the fluorescent protein produced over periods. In the beginning, we dilute the sample to six tubes with LB broth to 6ml in a ratio of 1:100. After dilution, we start to measure the optical density and fluorescence intensity. For each time, we take 200μl each time and plate it into a 96-well plate (costar, flat bottom with lid). Finally, we put the plate into CLARIOstar® Plus plate reader to measure OD and fluorescence. The parameter for fluorescence is shown below:

Table 1:The parameters for fluorescence

Parameter	Value
Method	Fluorescence intensity
Mode	Endpoint
Optic	Top optic
Microplate	SBS STANDARD 96
Focal height	Adjust every test run
Gain adjustment (Target value)	5 %
Excitation	503 ± 8 nm
Emission	537 ± 8 nm
Orbital Averaging (diameter)	4 mm
Speed and Precision	Precise

2. Results

A. Colony PCR and SDS PAGE

Figure 1: The result of PCR. In the first well is the 1kb DNA ladder, and the second well is our PCR product. Our target gene(BBa_I6060) is 1125 b.p.

Figure 2: The comparison of bacteria with eYFP+IPTG, eYFP-IPTG and pSB1C3 backbone

B. Optical Density and Fluorescence

After all the confirmation in part A, we started to measure the fluorescence and optical density of this fluorescence protein. After 18 hours of observation, we got the result, as shown below.

Figure 3: The results of the fluorescence intensity of eYFP over time

Figure 4: The relations between fluorescence intensity and time

Figure 5: The relations between fluorescence intensity and OD value

According to the results, in figure 3, the OD value of three kinds of samples (eYFP+IPTG, eYFP-IPTG, pSB1C3 backbone) all have similar growth curves, and the backbone grows faster because it has less growth pressure.

In figure 4, the fluorescence of eYFP+IPTG has a trend that grows in a constant speed, but the other cure, eYFP-IPTG, since there is no IPTG to open lac promoter, the growth seems to be restricted. Therefore we can conclude that and the trend is similar to the OD value, we guess it’s because it has a constitutive promoter, so the amount of protein will be highly related the lac promoter is really functioning.

In figure 5, the relation between optical density and fluorescence intensity of the sample eYFP+IPTG is 0.692, and it belongs to moderate correlated. Although it’s not entirely agreed with what we suspect, it’s still close to highly correlated, so we think the error here is just because of the mistakes we make while we measured the data.

II. Quorum sensing system

To accomplish the idea of visualization, we have already started the preliminary experiment. First, we cloned the quorum sensing system into the E. coli and conducted the functional test. Then, we co-transformed them with toxin genes. In future experiments, we will do functional tests of the co-transformed E. coli. After that, we will put the co-transformed E. coli together with different mutagens and accomplish furthermore progress to our project.

Quorum Sensing Gene

Cloning

The Figure 6 is the electrophoresis results of the PCR products with 1Kb marker on the left side and target gene on the right side. The lengths are labeled beside each band.

Figure 6: DNA electrophoresis of part BBa_K3256437 with BBa_K3256439.

As the figure we show above, we successfully cloned QS gene into the E. coli.

Functional Test

Then, we tested the function by measuring the fluorescence intensity and the optical density of the transformed E. coli together with controlled group. We put our sample into the CLARIOstar® Plus plate reader. For each sample, we applied triple replication in order to get more precise data.

Figure 7: EtBr mutagenic effect on E. coli growth with quorum sensing rfp expression

In the beginning, the fluorescence intensity remained relatively at low level. At about 150 minutes after IPTG induction, the fluorescence increased sharply and then remained in high intensity. The result was in accordance with our quorum sensing design. The sharp increase of fluorescence intensity occurred at O.D. value at approximately 0.2, which was expected to be too early. This phenomenon was observed in multiple tests. We expected that the promoter Plux was loose and expressed well with little concentration of AHL. For future work of integrating Quorum Sensing system in the bioassay, we may have to change the promoter or add operon for the tighter control.

Composite Parts

Cloning

After we had done the function test of toxin gene and the QS gene circuit, we co-transformed the two vectors into E. coli BL21(DE3).

Figure 8 was the electrophoresis results of the PCR products with the marker on the left side and target gene on the right side. The lengths are labeled beside each band.

Figure 8: A. DNA electrophoresis of part BBa_K3256440 and BBa_K3256437 with BBa_K3256439. B. DNA electrophoresis of part BBa_K3256443and BBa_K3256437 with BBa_K3256439.

We successfully co-transformed two different toxin genes respectively with QS gene into the E. coli. So, we will use them to do the following experiments.

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