Part:BBa_I6060

Promoter O_H Test R0010.E0432

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Team NCTU Formosa 2019 Characterization

Introduction

   To achieve the standard of characterization. This year, we measured the function of biobrick BBa_I6060, a kind of yellow fluorescent protein. In our plan, 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 typically. 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®.

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

Results


A. Colony PCR

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 time

   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 at 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.

Conclusion

   After all the experiments we did, we proved that the biobrick, BBa_I6060, we get in the kit could function normally inside E. coli BL21. It can successfully produce yellow fluorescence protein when IPTG is induced. We hope our experiment will help to improve the accessibility and comparability of reliable data in synthetic biology labs across the world. Thanks to iGEM for providing this opportunity to give back to the scientific community.