Lactate Detection
Biobrick Assembly
Figure 1 1%Agarose, 100V. Check the size of insert.
Length: pUCIDT(2705 bp)- J23118-LldR(1006bp)
Length: pUCIDT(2705 bp)- O1-J23118-O2-GFP(1235bp)
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 received our IDT plasmid DNA, the most important thing was to check the insert size was correct or not. We digested (EcoRI-PstI) it to check the size of plasmid. From Agarose gel result, we knew the length of 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 these agarose gel we known the size of backbone that we wanted was right. After knowing that, it is the time to assamble 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 finished assambling our parts, we did some function test, to find the relationship between GFP expression and lactate level.
Figure 4 Fluorescence of each lactate concentration depends on time.
We found that even though fluorescence in each concentration of lactate are floating, the spacing of each data are stable expect data 10 mM. It seems to be saturated when lactate concentration reaches 8 mM.
- (a)The fluorescence of each lactate concentration depends on time.
Figure 5 Fluorescence of different time depends on lactate level.
The interval 120 minute ≤ time ≤210 minute, the fluorescence of each concentration of lactate is similarity. Positive correlation exists between fluorescence and concentration of lactate on an interval 120 minute ≤ time ≤270 minute.
- (b)Fluorescence intensity of different time period depends on lactate level.
Figure 6 Combine Figure 4 and Figure 5 together.
We found that fluorescence level was high at the beginning. The general trend of fluorescence became pronounced and stable as time goes by.
- (c)It’s quite interesting that the fluorescence value always be very high at the beginning but fell as time passed by, even in negative control. The tendency of whole line is similar. And if we just see a single time point (b), a highly positive correlation between fluorescence and lactate level can be observed.
From figure 4~6, we found that there is a positive correlation between lactate concentration and fluorescence value. Below we took several points from 210 min and 270 min to create figures.
Figure 7 Fluorescence vs. Concentration of lactate at time=210 min
Figure 8 Fluorescence 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 fluorescence and lactate concentration between 2 mM and 8 mM of concentration of lactate. It shown that R-square reach 0.9957 at time 210 min and R-square reach 0.9965 at time 270 min. At time 270 min, R-square reach 0.9552 between 0 mM and 10 mM of concentration of lactate.
Summary
At first, we thought that the high level expression of CSP in the beginning is due to bad expression rate of LldR (inhibitor) and leakage of GFP. After 90 minutes, the LldR express was better so the fluorescence decrease. But we noticed that the negative control (E.coli that without fluorescence gene) tendency is similar with other data, we thought that maybe there is an intra system error lead this result.
Lactate detect
PCR result
Prove that we successfully transformed our plasmid into E.coli, DH5α.
a. pSBBS1C
b. pSBBS4S
Bacillus subtilis competence cell preparation and transformation results.
1. Growth curve of Bacillus subtilis strain 168
2. Transformation check for pSBBS1C
Since the amyE locus is knocked down, 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 the colonies when Lugol’s iodine is added.
Functional test results
According to the measurement of absorbance of wavelength 595 nm, we can confirm that CSP is’nt toxin to Bacillus Subtilis.
And for the measurement of the fluorescent plate reader (excitation: 485 nm, emission: 520 nm), we found out that the best amount of bacteria for measuring, the CSP conc. is at the value of OD600 = 0.2 after a series of experiment.
The intensity of fluorescence slowly drops after a sharp rise in a small amount of time, hence we can tell that the reaction starts right away as soon as CSP is added.
