Team:Shanghai Metro/Results
RESULTS
Overview:
We extracted plasmid vector Pus232
and ike/tke components from Pseudomonas putida
respectively and used restriction endonucleases to digest them for connection. At last, we constructed two
new plasmids: Pus232-ike2-tke2 and Pus232-ike4-tke4. Both plasmids have minor genetic differences, thus we
also proceeded with further tests to identify which one is more efficient at growing when the same optimal
amount of tetracycline is added.
1. Pus232 Electrophoresis
Figure 1. Gel electrophoresis of plasmids Pus232.
Channel 1~14: Purified plasmid Pus232 from 1~14 E. coli DH5α cultures.
This step is used to test if the plasmids Pus232 extracted from the E. coli DH5α
are successful and could be used to do double enzyme digestion later in the process.
Based on the three types of structures plasmid may have, we collect the supercoiled
bands. Channel 2, 5, 6, and 7 are the best bands and are selected for making double enzyme digestion.
Channel 3, which has a high concentration of plasmid, was once selected but failed.Therefore, channel 2, 5,
6, and 7 plasmids are selected to do double enzyme digestion of BamHI and XbaI for 2 hours.
Channel 3 might have undergone too long of a P2 cracking phase, which leads to the
fracture within the DNA. The DNA secreted contains groups of open circular DNA , showing as the bright white
color in channel 3, but less circular plasmid DNA that we are targeting to collect.
2. ike2/4 Electrophoresis
Figure 2. Gel electrophoresis of ike2 and ike4 PCR products.
Channel 1: ike2 electrophoresis failed maybe because bacteria added is too much.
Channel 2: ike2 succeeded
Channel 3: ike2 electrophoresis failed maybe because bacteria added is too much.
Channel 4: ike4 succeeded
Channel 5: ike4 succeeded
Channel 6: ike4 succeeded
Channel 7: ike4 succeeded
This step is used to check if the ike2 and ike4 extracted from Pseudomonas putidas
are successful and could be used to do double enzyme digestion later in the process.
PCR clean-up ike2 and ike4 DNA fragments to do double enzyme digestion of BamHI and XbaI overnight.
Channel 1 and 3 failed the test. One possible explanation could be that the
bacteria added into the PCR solution is too much.
3. Pus232 Double Enzyme Digestion
Figure 3. Gel electrophoresis of Pus232 double enzyme digestion products.
Channel 1&2: Pus232 control group
Channel 3~6: Products of Pus232-BamHI+XbaI Double Enzyme Digestion
Channel 7~10: Products of Pus232-BamHI+XbaI Double Enzyme Digestion
Channel 3~10 have shorter bands after the double enzyme digestion, and all of them
are successful. Gel clean-up the product of Pus232-BamHI+XbaI double enzyme digestion to obtain
Pus232-backbone. Clean-up the product of ike2 and ike4-BamHI+XbaI overnight double enzyme digestion to
obtain ike2-fragment and ike4-fragment.
T4 DNA ligase is used to connect Pus232-backbone with ike2-fragment and
ike4-fragment overnight separately.
4. Pus232-ike2/4 sequencing analysis
Figure 4. Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of
Pus232-ike2.
Figure 5. Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of
Pus232-ike4.
The sequencing results show that both Pus232-ike2 and Pus232-ike4 are constructed
successfully.
5. Electrophoresis of Pus232-ike2/4 Enzyme Digestion
Figure 6. Gel electrophoresis of Pus232-ike2, Pus232-ike4 enzyme digestion products.
Channel 1~4: Products of Pus232-ike2-SacII single enzyme digestion, succeed, cut
the target bands for gel clean-up.
Channel 5~8: Products of Pus232-ike4-SacII single enzyme digestion, succeed, cut
the target bands for gel clean-up.
Channel 9~12: Products of tke4 PCR, contains some unneeded bands, cut the 4k+ bands
for gel clean-up.
6. Electrophoresis of tke2/4
Figure 7. Gel electrophoresis of tke2 and tke4 PCR products.
Channel 1~3: tke2 KOD, succeed, bands size are correct and unitary, cut for gel
clean-up.
Channel 4~6: tke4 KOD+DMSO, succeed, contains some unneeded bands, cut the 4k+
bands for gel clean-up.
7. Recombination of the Linearized Pus232-ike vectors and tke inserts
The tke insert and the linearized Pus232-ike vector, with overlapped sequences of
15 bp on both 5’- and 3’-end, respectively, are mixed and incubated with recombinase Exnase II at 37°C for
30 min. Pipet 8 μl of the recombination products to 80 μl of the E.coli DH5α competent cells for
transformation. Pipet 4 μl of the control group Pus232 and Pus232-ike2 to 40 μl of the E.coli DH5α competent
cells for transformation.
8. Pus232-ike4-tke4 Transformation Plates
Control group E.coli DH5α/Pus232 and E.coli DH5α/Pus232-ike2 show blue strains,
which are desired results because without the tke presence, lacZ protein in Pus232 won’t be replaced, and
X-Gal we added under the catalysis of lacZ protein, blue products will be produced, thus the strain
appearing blue(Fig. 8 left and middle). However, the white single strain may be microbial contamination
(Fig. 8 left).
Figure 8. Transformation plates of recombination reaction.
Experimental group Pus232-ike4-tke4 had a white single strain when the picture was
taken (Fig. 8 right). The result could be satisfactory because tke4 replaced lacZ protein in the plasmid,
stopping it from expressing thus the strain won’t demonstrate any blue color.
9. Functional Test
We first identified if we have correct strains with naked-eye observation, with
tke4 present, the strain color should be white, blue strains may be undesired organisms or microbial
contamination. Then we took a single strain for OD600 Testing to further determine the range of optimal
tetracycline amount.
OD600 Testing results are below:
With the result, we can generally determine the appropriate tetracycline induction
concentration range is
0-0.1μg/mL or more, only less than 1μg/mL. Due to the large concentration range selected in the preliminary
test, it is impossible to determine the effect of the induced concentration between 0.1-1μg/mL. We perform
further tests to keep a smaller range until we find the optimal tetracycline amount.
The result also proved the effect of tetracycline alone, the graphs below show that more tetracycline in
the. control group Pus232 will suppress the growth of strains. As for our modified plasmid strain, the curve
is roughly similar to the parabola concave down, which has a maximum or optimal amount in this case.
Figure 9. Biomass Concentration curve of Pus323 Strain with tetracycline
Figure 10. Biomass Concentration curve of Pus323-tke4-ike4 Strain with tetracycline
After having a general idea of the best concentration of tetracycline, we set up
different time knots to measure the concentration of strains, each with a two hours interval. Similarly, the
OD600 testing will help us to determine the growth of strains. The results of the test are below.
Despite being less effective than 200µg/L concentration amounts at early hour
measurement, eventually, concentrations at the range of 300-500µg/L are more effective. The results also
proved that adding tetracycline to ike4-tke4 strain will boost the growth of strain, while the effect to
Pus232 strain alone is less obvious. This test is more thorough than our first one, and the induced
concentration of 100-1000μg/L is more detailed. Now it determines that the optimal concentration of
tetracycline is 300µg/L to 500µg/L.
Figure 11. Biomass Concentration curve of Pus323-tke4-ike4 Strain with tetracycline against
hours
Figure 12. Biomass Concentration curve of Pus323 Strain with tetracycline against hours
Future approach:
One disadvantage of our technique is that this only applies to the E.coli bacteria
since the modification is done on the E.coli plasmid. This prevents us from cooperating with companies whose
bacteria is not modified on E.coli. Therefore, possible future approaches could be to discover how we can
protect the patent of bacterias other than E.coli. We may transfer our modified plasmid into other bacteria
strains or design a new plasmid for other types of bacteria.