Difference between revisions of "Team:UI Indonesia/Improve"

As a contribution of iGEM UI 2018 team to improve existing parts in iGEM Registry, we decided to assemble “Improved BFP” (BBa_K2607002, Figure 1) as improvement of mTagBFP (BBa_K592100) and B0034-BFP (BBa_K592024). Our established part consists of these following features:

Figure 1. Original designed “Improved BFP” (BBa_K2607002) gBlock we ordered from Integrated DNA Technologies, Inc. (IDT) with length of 1151 basepairs (bp). Note that extra 42 bases in the upstream and 41 bases in the downstream are BioBrick prefix and suffix and for polymerase chain reaction (PCR) amplification purpose. The part itself consists of 1068 bp. Range number in red indicates the position of each component. Rbs = ribosome binding site.

• lacI regulated promoter (BBa_R0010) in the upstream
• Double terminator (BBa_B0010 and BBa_B0012) in the downstream
• SalI restriction site between the promoter and ribosome binding site
• NdeI restriction site between the ribosome binding site and coding sequence
• Modified blue fluorescence protein coding sequence to eliminate SalI restriction site in the middle without altering amino acid sequence.

This part can be used as reporter when combined with other parts. Because this part has its own promoter, ribosome binding site, and terminator, addition of these components is basically unnecessary. In addition, Appropriate restriction enzyme (NdeI or SalI) can also be used to replace the promoter or ribosome binding site with the desired one.

Figure 2 shows the original plan of how we conduct the experiment with our established and existing parts.

Figure 2. Original plan workflow with newly-established (BBa_K2607002) and existing (BBa_K592024) parts of BFP.

Part I: Gel Electrophoresis Confirmation

Upon receiving our “Improved BFP” gBlock from IDT, we performed PCR to amplify it until sufficient quantity (about 100 ng/uL). We then created two restriction digestion reactions, one with NdeI restriction enzyme and one with SalI restriction enzyme. Each reaction comprised of 100 uL PCR-amplified “Improved BFP” gBlock, 6 uL restriction enzyme, 15 uL CutSmart^® restriction buffer, and 29 uL nuclease-free water. The reaction was subsequently incubated for four hours at 37^oC incubator. The digestion products were then run for electrophoresis in 1% agarose gel with tris-acetic acid-EDTA (TAE) buffer 1x, power supply 50 volt for 70 minutes. Visualization was performed with Bio-Rad Gel Doc^TM XR+ Gel Documentation System.

Part II: Ultraviolet (UV) Check

First, we acquired BBa_K592024 by transforming wild-type Escherichia coli K-12 with plasmid in well 17C of Distribution Kit Plate 1, isolating the plasmid, and performing restriction with EcoRI and PstI restricton enzymes. Our “Improved BFP” and BBa_K592024 were separately cloned into plasmid pQE80L using EcoRI and PstI according to our laboratory protocol. The resultant ligation products were subsequently transformed into wild-type E. coli K-12, along with empty pQE80L as control. Transformation products were spread into Luria-Bertani (LB) agar medium containing ampicillin with ratio 1000:1. On the following day, the fluorescence was assessed qualitatively under ultraviolet (UV) light.

Part III: Fluorescence Assay

Fluorescence assay was conducted according to 5^th InterLab protocol with some modifications. From each transformation plates, two colonies were picked and let to be grown in LB medium with ampicillin (1000:1). Two colonies of E. coli containing empty plasmid pQE80L were also inoculated for control. After 18 hours, each overnight culture was diluted until optical density at 600 nm (OD₆₀₀) of 0.02 and final volume of 12 mL in 50 mL centrifuge tube covered with foil. At t = 0 hour, 1 mL from each tube was sampled to be measured for its OD₆₀₀ and fluorescence. The measurement was conducted with GloMax^® - Multi Detection System with “UV” settings: 365 nm excitation and 410-460 nm emission. The data obtained were recorded and analyzed in Excel. The rest of dilutions were incubated in shaker under 37^oC, 220 rotations per minute (rpm). We repeated the same procedure after t = 6 hour and overnight (t = 14 hour) incubation.

For complete raw data of calibrations (in Part I) and Abs₆₀₀ and fluorescence measurement (in Part II), please refer to our Excel file attached here.

Part I: Calibration

Table 1. OD₆₀₀ reference point results.

When measured with our plate reader, mean Abs₆₀₀ of LUDOX CL-X was 0.156 and mean Abs₆₀₀ of ddH₂O was 0.096 (Calibration #1). By subtracting the means, we obtained the corrected Abs₆₀₀ value was 0.059. After dividing the reference OD₆₀₀ (0.063, provided in Excel file) with corrected Abs₆₀₀, we found that the conversion factor was 1.059 (Table 1). In other words, when we wanted to transform Abs₆₀₀ value measured by our plate reader into OD₆₀₀, we must multiply the Abs₆₀₀ data with 1.059.

Figure 11. Particle standard curve results.

Particle standard curves we obtained after conducting Calibration #2 are shown in Figure 11. The equation for particle standard curve was y = 3*10^-9x + 0.078 (R² = 0.9945) and for particle standard curve in log scale was y = 6*10^-4x^0.3536 (R² = 0.8819), with x is particle count per 100 uL sample and y is Abs₆₀₀. As shown in the graphs, the values measured form a straight line and the slope is close to 1:1 until it saturates at low concentration of microspheres.

At medium-high concentrations of microspheres (i.e. from 7.35*10⁶ until 1.18*10⁸ particles/100 uL), we found the mean particle concentration after divided by their respective Abs₆₀₀ was 2.08*10⁸ particles/100 uL sample. We would later use this value to estimate molecules of equivalent fluorescein (MEFL) per particle in Part II (i.e. in this case, the particles were bacterium cells, and microspheres were used as a representation of the cells). We used medium-high concentrations value because they are less likely to be affected by pipetting error or saturation (the same principle applies to fluorescein which will be discussed later).

Figure 12. Fluorescein standard curve results.

Our results for fluorescein standard curve after conducting Calibration #3 are shown in Figure 12. The equation for fluorescein standard curve was y = 139561x + 18723 (R² = 0.9957) and for fluorescein standard curve in log scale was y = 176245x^0.8822 (R² = 0.9985), with x is fluorescein concentration in uM and y is fluorescence in arbitrary units (a.u.). As shown in the graphs, the values measured also form straight line and the slope is close to 1:1.

At medium-high fluorescein concentrations of fluorescein (i.e. from 0.31 until 5 uM), we found the mean concentration of fluorescein after divided by their respective fluorescence level was 5.84*10^-6 uM/a.u. When this mean value was converted into MEFL (MEFL/uM fluorescein value is 6.02*10¹², provided in the Excel), we found that MEFL per arbitrary unit was 3.52*10⁷. We would later use these values to convert raw data obtained in Part II into fluorescence per OD (uM fluorescein/OD) and fluorescence per particle (MEFL/particle).

Part II: Cell Measurements

Table 2. Mean Abs₆₀₀ for 1:10 diluted transformed devices. *= calculated by 240/(∆Abs₆₀₀*10) where 240 is target Abs₆₀₀*target volume (12mL) and 10 is dilution factor used. TD = test device

First, we created samples of 1:10 dilution from each transformed device and checked for their respective Abs₆₀₀ by creating two replicates from each diluted sample. The absorbance result is shown in Table 2, second column. Using the dilution formula, we obtained volume of each initial culture to be added into fresh LB + Cam to target Abs₆₀₀ approximately 0.02 and final volume of 12 mL (Table 2, fourth and fifth column). To ensure Abs₆₀₀ of the newly-made dilutions were about 0.02, we performed Abs₆₀₀ measurement along with their fluorescence at t = 0 hour. The rest of dilutions were then incubated and after 6 hours, their Abs₆₀₀ and fluorescence were measured again.

Figure 13. Fluorescence per OD result.

Figure 14. Fluorescence per particle result.

Results of fluorescence per OD and per bacterium cell particle are shown in Figure 13 and Figure 14, respectively. When compared, both graphs look similar in pattern, however fluorescence of tested devices were differed from each other. Therefore, it can be implied that some promoters may have different activity in expressing its downstream genes (in this case, GFP). The data at t = 0 hour suggested that promoter J23101 (Test Device 1), J23100 (Test Device 4), and J23104 (Test Device 5) have higher activity in expressing GFP, resulting in higher fluorescence per OD and per particle compared to positive control. Furthermore, fluorescence observed in Test Device 2 and 6 were comparable with positive control, implying that promoter J23106 (Test Device 2) and J23116 (Test Device 6) have similar activity in expressing GFP with promoter J23151 (positive control). On the other hand, there was no fluorescence observed in Test Device 3 (similar with negative control that does not have GFP construct). It is likely that promoter J23117 (Test Device 3) failed to initiate GFP expression.

At t = 6 hour, declines in fluorescence per OD and per particle were observed in positive control, Test Device 2, Test Device 4, and Test Device 6, compared with t = 0 hour. A probable explanation for this phenomenon is that GFP expression mediated by promoters carried by these devices is limited by cell density (i.e. no additional GFP expression when cells in a sample has reached a certain density), or cells in the culture underwent cessation. Therefore, when total fluorescence was divided by the cell number in given sample, fluorescence per OD and per particle will be decreased. Exception for Test Device 1 and 5, increase in fluorescence per OD and per particle were observed, suggesting that promoter J23101 and J23104 may be strong promoters that actively express their downstream genes despite high density of cells.

Part III: Colony Forming Units (CFUs)

Table 3. Mean Abs₆₀₀ for 1:8 diluted transformed devices. * = calculated by 100/(∆OD₆₀₀*8) (in uL), where 100 is target OD₆₀₀ (0.1)*target volume (1 mL) and 8 is dilution factor used.

Table 4. OD₆₀₀ measurement from starting samples.

We created 1:8 dilution from two positive control samples and two negative control samples (obtained after overnight incubation in Part II) and measured for their Abs₆₀₀ (Table 3, second column). Next, we converted Abs₆₀₀ data into OD₆₀₀ by multiplying them with 1.059 (i.e. conversion factor obtained from Part I). From this, we calculated for overnight culture volume to be added into fresh LB + Cam to target OD₆₀₀ 0.1 and final volume 1 mL using dilution formula (Table 3, fifth and sixth column). We then checked Abs₆₀₀ from newly-made dilutions whether their OD₆₀₀ were around 0.1 after subtracted by blank. The results is shown in Table 4.

Figure 15. Representation of dilutions grown in LB + Cam agar. (Top row, from left to right) 8 x 10⁴, 8 x 10⁵, and 8 x 10⁶ dilution of positive control. (Bottom row, from left to right) 8 x 10⁴, 8 x 10⁵, and 8 x 10⁶ dilution of negative control.

Table 5. Colony count results. TNTC = too numerous to count (>300 colonies). N/A = not calculated due to out of optimal 50-200 colony count range.

We performed serial dilution as suggested in iGEM 2018 InterLab Protocol from the dilutions and the results is shown in Figure 15 and Table 5. Based on plates with optimal colony count (i.e. 50-200 colonies), we found that estimated CFU concentration per 0.1 OD₆₀₀ of positive control culture 1, positive control culture 2, negative control culture 1, and negative control culture 2 were approximately 1.05 x 10⁸, 1.25 x 10⁸, 6.16 x 10⁷, and 6.69 x 10⁷ CFUs/mL, respectively (Table 5, eighth column).

Table 6. Manual conversion of fluorescence per OD into fluorescence per CFU, compared with fluorescence per particle.

Finally, we tried to manually convert our obtained fluorescence per OD (in Part II) into fluorescence per CFU using estimated CFU concentration per 0.1 OD₆₀₀ (in Part III), and compared them with our obtained fluorescence per particle (in Part II). The analysis for t = 0 hour can be seen in Table 6 (detail calculation can be found in our Excel file). We found that our calculated fluorescence per CFU were different with fluorescence per particle. Therefore, our results suggested that the benefit of using direct method in expressing total cell number such as colony forming units (CFUs) than OD in reporting fluorescence per cell remains doubtful. However, to validate our findings, additional data from another teams are necessary.