SECTION II: Protocol

Introduction: There are no spectrophotometers – neither UV nor fluorescent – in Sumbawa island. Thus our project uses mobile phone camera to obtain data and quantify using customized software. Among fluorescent proteins, mRFP showed strong color visible to naked eye, thus we chose it as the report of measurement in our project. However, this report was made in the request of iGEM 2014 Committee using GFP to show the possibility on using our system for the Measurement Interlab Study. In this study we used different device/part although it has the same GFP gene, aside from which was already been designated by the Committee. We did it because the objective was to show whether GFP is measurable or not in our system.

Through this activity, people's understanding of Sumbawa honey is also increasing. We introduce to the public some information about Sumbawa honey such as how to distinguish between genuine and fake honey, a tool used to measure the levels of glucose in honey, until any product that can be produced made from honey. We hope this activity could expand the knowledge and society pride of Sumbawa honey. We also hope that our project will make people aware of the importance of maintaining the natural resource and pride of this region. When the awareness is growing, we believe the existence of honey as the pride of Sumbawa could be maintained.

Materials: E. coli BL21(DE3), and E. coli BL21(DE3) / BBa_I13522

Methods: Four glass tubes were prepared to cultivate overnight E. coli in 2 mL LB medium, in room temperature using shaker. The content of each tubes were as follow:

• Tube-1. E. coli BL21(DE3) only. No antibiotics. Used as control.
• Tube-2. E. coli BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final).
• Tube-3. E. coli BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final); Auto induction reagents (Studier, Protein Expression and Purification 41, 207-234, 2005).
• Tube-4. E. coli BL21(DE3) / BBa_I13522; Chloramphenicol 34 ug/ml (final); Glucose 200 mM.

After one night of cultivation, tubes were collected, then photos were taken using pocket digital camera, and color of the medium were measured using free application available on Google Play named ColorMeter, an application developed by vistechprojects.com. The mobile phone used was Samsung GT-P5200 having 3.15 MP camera. The medium were centrifuged (6,000 rpm, 10 min.) in 1,5 ml polypropylene tube to obtain cell pellets, which were used to detect GFP fluorescence above a blue transilluminator (Pearl Biotech).

SECTION III: Results and Discussions

In this study we used BBa_I13522, which already available in our lab, instead of BBa_I20260 which is designated as device no. 1 by Measurement Interlab Study Committee. As shown in Figure 1, both devices have the same RBSs, genes and terminators, except promoters. Because our intention here is to prove whether our system using mobile phone camera is suitable for GFP measurement or not, we use BBa_I13522 for convenient reason.
Figure 1. Vector maps of BBa_I13522 (left) and BBa_I20260 (right)

In Figure 2, it is showed that medium from 4 tubes showed similar color, although tube no. 3 has more green. Tube no. 1 and 2 are negative controls because they contain E. coli BL21(DE3) only, and E. coli BL21(DE3) / BBa_I13522 without expression reagent to induce GFP production, respectively. Tube no. 3 contains E. coli BL21(DE3) / BBa_I13522 with GFP was over expressed due to addition of auto induction reagent. On the other hand, glucose was added to tube no. 4 to repress possible leaky expression of GFP through the mechanism of catabolite repression.
Figure 2. Color of an overnight LB medium grew with E. coli under different condition. 1) E. coli BL21/DE3; 2) E. coli BL21 (DE3)/Bba_I13522; 3) E. coli BL21(DE3)/BBa_I13522 + autoinduction reagent; 4) E. coli BL21(DE3) + glucose 200 mM.

ColorMeter program installed in mobile phone, detected RGB values, we then converted the values to luminance value to obtain only the brightness of color (http://www.scantips.com/lumin.html; accessed on Sep. 24th, 2014). The formula used is:

Luminance value = (R x 0.30) + (G x 0.59) + (B x 0.11)

Two measurements of RGB values of the tubes from different angles to correct possible lighting differences, and the calculation of luminance values are given in Table 1 and Figure 3 below.

Table 1. RGB and Luminance value obtained by ColorMeter program
Figure 3. Luminance values of an overnight LB medium grew with E. coli under different condition. 1) E. coli BL21/DE3; 2) E. coli BL21 (DE3)/Bba_I13522; 3)E. coli BL21(DE3)/BBa_I13522 + autoinduction reagent; 4)E. coli BL21(DE3) + glucose 200 mM.

Expression of GFP in theory should make the color of medium brighter thus luminance value will be lower compared to E. coli which did not express GFP. Tube no. 1, and 4 has higher luminance value than tube no. 3, which is as expectation. But tube no. 2 showed low luminance value too. The reason may be because the color differences are too small (luminance value range < 5), thus accuracy of the results was low. In our project using mRFP (data not shown), the difference of luminance value between expressed and non-expressed mRFP were about 50.

Finally to validate the expression of GFP, we used the only equipment available in our Lab, which can visualize specifically fluorescent protein including GFP ((http://en.wikipedia.org/wiki/Green_fluorescent_protein; accessed on Sep. 24th, 2014). Two observations were made, in liquid and solid states. As shown in Figure 4 (top), observation of the culture medium showed that tube no. 3 has bright yellow color compared to other tubes. The color should come from GFP expressed by E. coli BL21(DE3)/BBa_I13522 cultured with auto induction reagent. However, the color was not very significant. Thus, cells were pelleted by centrifugation and once again observed. As shown in Figure 4 (bottom), tube no. 3 showed much bright yellow color compared to other tubes. This result concluded that this tube really containsE. coli BL21(DE3)/BBa_I13522 expressing GFP.

Figure 4. Observation of GFP in liquid medium (top) and pellet (bottom) by transilluminator 1) E. coli BL21/DE3; 2) E. coli BL21 (DE3)/Bba_I13522; 3) E. coli BL21(DE3)/BBa_I13522 + auto induction reagent; 4) E. coli BL21(DE3) + glucose 200 mM

In conclusion, our measurement system using mobile phone camera could not detect GFP expression in liquid medium because of small differences observed by naked eyes. Thus, we used mRFP instead for our iGEM project.