Figure 1. 1% agarose, 100V.
Check the size of insert.
- Length: pUCIDT(2705 bp)- J23118-LldR(1006 bp)
- Length: pUCIDT(2705 bp)- O1-J23118-O2-GFP(1235 bp)
- Sample 1: pUCIDT-J23118-LldR without digestion
- Sample 2: pUCIDT-J23118-LldR digested with EcoRI and PstI
- Sample 3: pUCIDT-O1-J23118-O2-GFP without digestion
- Sample 4: pUCIDT-O1-J23118-O2-GFP digested with EcoRI and PstI
After our IDT plasmid DNA arrived, the most important thing was checking the insert size was correct or not. We digested (EcoRI-PstI) it to check the size of plasmid. From the agarose gel result, we can confirm the inserts are correct.
Figure 2. 1% agarose, 100V.
Check the size of pSB1A2.
- Length: pSB1A2(2079 bp)-E0032(762 bp) digested with EcoRI and PstI
- Sample 1: pSB1A2-E0032 without digestion
- Sample 2: pSB1A2-E0032 digested with EcoRI and PstI
From this agarose gel we can confirm the size of backbone that we wanted was right. Next, it is the time to assemble them together !!
Figure 3. 1% agarose, 100V.
Check the size of assemble
[pSB1A2 (2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR (2241 bp)].
- Sample 1: [pSB1A2(2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR(2241 bp)] digested with EcoRI and PstI
- Sample 2: [pSB1A2(2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR(2241 bp)] digested with EcoRI and PstI
- Sample 3: [pSB1A2(2079 bp)]-[O1-J23118-O2-GFP-J23118-LldR(2241 bp)] digested with EcoRI and PstI
Lactate detect function test
After finish assembling our parts, we did some function tests, to find the relationship between GFP expression and lactate level.
Figure 4. Fluorescent intensity of different lactate concentration depends on time.
We found that even though fluorescent intensity in different concentration of lactate floats, the spacing of each data are stable expect data 10 mM. It seems that lactate concentration is saturated when it reaches 8 mM.
- (a) The fluorescent intensity of different lactate concentration depends on time.
Figure 5. Fluorescent intensity of different time depends on lactate level.
The interval 120 minute ≤ time ≤210 minute, the fluorescent intensity of different concentration of lactate is similar. Positive correlation exists between fluorescent intensity and concentration of lactate at the interval 120 minute ≤ time ≤270 minute.
- (b) Fluorescent intensity of different time period depends on lactate level.
Figure 6. Combine Figure 4 and Figure 5 together.
We found that fluorescent level was high at the beginning. The general trend of fluorescent level became pronounced and stable as time goes on.
- (c) It’s quite interesting that the fluorescent level had always been very high at the beginning but fell as time passed by, even in negative control. The tendency of the whole line is similar. And if we just look at a single time point (b), a highly positive correlation between fluorescent intensity and lactate level can be observed.
From Figure 4~6, we found that there is a positive correlation between lactate concentration and fluorescent level. We took several points from 210 min and 270 min to create Figures which are shown below.
Figure 7. Fluorescent intensity vs. Concentration of lactate at time=210 min
Figure 8. Fluorescent intensity vs. Concentration of lactate at time=270 min
We can see that the error bar is large in low concentration of lactate but is small in high concentration. We also find that it seems to have a high positive correlation between fluorescent intensity and lactate concentration between 2 mM and 8 mM of concentration of lactate. It shown that R2 reach 0.9957 at time 210 min and R2 reach 0.9965 at time 270 min. At time 270 min, R2 reach 0.9552 between 0 mM and 10 mM of concentration of lactate.
At first, we thought that the high level expression of GFP in the beginning is due to the bad expression rate of LldR (inhibitor) and the leakage of GFP. After 90 minutes, the expression of LldR was better and the fluorescence decreased. But we noticed that the negative control (E.coli that without fluorescent gene) tendency is similar with the other data, we thought that maybe this was caused by an intra-system error.
Prove that we successfully transformed our plasmid into E.coli, DH5α.
Figure 9. PCR result for pSBBS1C.
The DNA size we expect to show up is 2563 bp, hence from the electrophoresis gel photos above, we can confirm that the plasmid DNA in well number 1, 3, 4, 8, 9, 10, 11, 13, 16, 19, 21, 24, 25, 27, 28 are those we want, and the corresponding colonies are those successfully transformed.
Figure 10. PCR result for pSBBS4S
The DNA size we expect to show up is 1344 bp, hence from the electrophoresis gel photos above, we can confirm that the plasmid DNA in all wells, except well number 5, 17, 18, 31, are the ones we want, and the corresponding colonies are those successfully transformed.
The results of Bacillus subtilis competence cell preparation and transformation.
1. Growth curve of Bacillus subtilis strain 168
Figure 10. Growth curve of B. subtilis strain 168.
2. Transformation check for pSBBS1C
Since the amyE locus is knocked out, the transformed Bacillus subtilis won’t be able to digest starch. Hence, when it is patched onto starch plate and placed overnight, there shouldn’t be a bright orange surrounding around the colonies when Lugol’s iodine is added.
Figure 11. Starch plate test. Left: B. subtilis carry plasmid pSBBS1C; Right: B. subtilis stain 168.
Functional test results
According to the measurement of absorbance of wavelength 595 nm, we can confirm that CSP isn't toxic to Bacillus Subtilis.
Figure 12. Growth curve of B. subtilis in CSP fluorescence function test. Initial OD600=0.2.
We measure fluorescence (excitation: 485 nm, emission: 520 nm) in different concentration of CSP and in different initial value of OD600. Fluorescence is measured every 40 minute using the 96 well plate.
Figure 13. Fluorescent intensity vs. Concentration of CSP at different time, initial OD600=0.05
Figure 14. Fluorescent intensity vs. Concentration of CSP at different time, initial OD600=0.1
Figure 15. Fluorescent intensity vs. Concentration of CSP at different time, initial OD600=0.2
Figure 16. Fluorescent intensity vs. Concentration of CSP at different time, initial OD600=0.4
We found out that the best bacteria density for measuring, the CSP concentration is at the initial value of OD600 = 0.2 after a series of experiment.
The intensity of fluorescence slowly drops after a sharp rise in a short time, hence we can tell that the reaction starts right after CSP is added.
The fluorescence emitted at OD600 0.2 has a positive correlation with the conc. of CSP added. However, the fluorescence emitted by Bacillus subtilis is lower when a higher concentration of CSP is added, which doesn’t correspond to our expectation. And we are still figuring out what’s going on!