Team:KU Leuven/Research/Results
Results
Double Knockouts
The Centre of Microbial and Plant Genetics (KU Leuven) provided us with three E. coli K-12 strains with each one representing the knockout for the genes tar, tsr or cheZ. The kanamycin cassette of the tar knock-out strain was removed by the enzyme flippase on pCP20. This excision was checked by PCR. The original knock-out strain of tar was used as a positive control and a band at 1232 bp is expected. If the cassette is removed, a band at 438 bp is visible. Ten colonies were tested and all have lost their cassette (Figure 1).
Figure 1 Excision of the kanamycin cassette of the knock-out of tar.
As a positive control, the original knock-out of tar was used which should show a band at 1232 basepairs. If the kanamycin cassette is removed, a band at 438 bp will be visible. Besides, a 1 kb Plus DNA ladder of GeneRuler was used.
The PCP20 plasmid contains a temperature sensitive origin of replication. To remove this plasmid, the colonies were grown overnight at 42°C. PCP20 is resistant to ampicillin, this characteristic is useful to verify the removal of the plasmid. Single colonies were streaked on one LB plate with and one without ampicillin. Figure 2 proves that the PCP20 plasmid is removed in all plasmids.
Figure 2Test to prove that the ampicillin resistant PCP20 plasmid is removed. The left plate contains ampicillin, the right plate contains no antibiotic.
We kindly received lysate from Oscar Torres. Our donor strains (ΔcheZ and Δtsr) were infected with this lysate. In figure 3, the plaques as result of the infection is visible. Some of the plaques will contain DNA of ΔcheZ and Δtsr due the sloppy packaging mechanism of the phage P1.
Figure 3Plaques after infection of our donor strains (ΔcheZ and Δtsr).
The lysate was plated out on LB plates as control. No colonies are visible in figure 4, this means that are lysate is not contaminated by cells.
Figure 4Control to check if the lysate of ΔcheZ and Δtsr is not contaminated with cells.
The plaques of the acceptor strains were extracted and different amounts of lysate were used to infect our donor strain (Δtar). The result was plated out on kanamycin plates to select the right colonies (Figure 4).
Figure 5
Infection of the acceptor strain (Δtar) by lysate originating from donor strains (ΔcheZ and Δtsr)
Our Tar knock-out cells without the kanamycin cassette were also plated out on kanamycin as a control. In figure 5 is visible that there is no growth.
Figure 6
Tar knock-out cells without the kanamycin cassette were plated out on kanamycin plates as control.
Different colonies were screened to confirm the knock-out in cheZ. If the cassette is not there, a band should show at 863 bp. If the cassette is still there, there should be a band at 581 bp. As a positive control we used a knockout Tar strain which does not contain the kanamycin cassette anymore.
Figure 7
PCR to check that cheZ is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.
To confirm that the knock-out in tsr was successful, a PCR was performed. A knock-out in tsr should give a band at 1304 bp while the original gene should give a band at 1964 bp. As positive control, a knock-out in tar which lost the kanamycin cassette was used.
Figure 8
PCR to check that tsr is knocked out in Δtar. The positive control is a knock-out in tar which lost the plasmid.
Especially in the new constructed ΔtarΔcheZ, the chance exists that the knock-out in tar is undone due the sloppy packaging mechanism of the phage P1. Therefore, the knock-out in tar is checked again by PCR (see figure 9). The first positive control (c1) is a tar knock-out who has lost the kanamycin cassette. As a second control (c2), a cheZ knock-out strain was used. The gel of figure 8 shows that all our ΔtarΔtsr strains are ok, while only two of the ΔtarΔcheZ are still ok.
Figure 9
PCR to check the knock-out in tar after the P1 transduction
Finally, the all the genes of the operon containing cheZ were checked by PCR (see figure 10). As positive control the tar knock-out which does not contain the plasmid anymore was used, but each time with the primers corresponding to the checked gene. The negative control contains the mastermix but without template.
Figure 10
PCR to check the operon of ΔtarΔcheZ
Motility Test
The cheZ knock-outs are not able to swim anymore. Therefor we performed a phenotypical motility test. The result is visible in figure 11.
Figure 11
Motility test of our cheZ knock-out
Gibson Assembly Method
Method 1
The first Gibson assembly was performed using only gBlocks without a vector. Since the T5 exonuclease cleaves nucleotides from 5’ to 3’, an extra fill-in step was added after the 1 hour incubation at 50°C. Appropriate primers were added together with Phusion DNA polymerase and T4 DNA ligase to recreate the blunt insert fragment. The ligated fragments were checked by PCR and both reactions showed a band at the expected height (Figure 12). The 1-2-3 reaction however showed a lot of aspecific bands. The 5-6 fragment was stored untill further use and the 1-2-3 fragment was then digested to be directionally cloned in a pSB1C3 backbone and transformed in electrocompetent E. cloni cells.
Figure 12
Positive colonies of 1-2-3 were further checked by colony PCR. Of this check, only one colony seemed to have the right insert (Figure 13). Restriction digestion was however negative (Figure not shown).
Figure 13
Method 2
Because our method was aberrant compared to the IDT protocol, a vector with correct overhangs was PCR amplified. This vector was used in a one-reaction Gibson assembly. After transformation, liquid cultures were grown from which the plasmid DNA was isolated. Restriction mapping was performed (Figure 14) and showed presence of only pUC19.
Figure 13
Even after digestion with DpnI, pUC19 kept appearing (Figure ?).
Figure 14
To optimize the vector PCR, pUC19 was first linearized using the unique restriction enzyme XbaI. Again, the PCR product was digested with DpnI. This plasmid was also transformed as a negative control and did not show any positive colonies. The plates that should contain the insert did show positive colonies. These were further analysed and
OHHL detection
In the first step, the Chromobacterium violacein CV026 was grown with different concentrations of OHHL. The C. violacein CV026 was added to the mixtures at an OD of 1.11. Our cells were grown for 24 hours in air-lid culture tubes at 30 °C in a shaking incubator (200 rpm). In figure 1 is clearly visible that a violet pigment is produced.
Figure 1
Culture tubes of inoculated Chromobacterium violacein CV026 with different amounts of OHHL. click to enlarge
First the OD of our cultures were measured in a cuvette (1 cm). Then the violacein is isolated from the cells by centrifugation, resuspension in DMSO and a second centrifugation.
Figure 2
Violacein will be removed out of our samples by centrifugation and resuspension in DMSO. Click to enlarge
After isolating violacein from our samples, 200 µL was pipetted into a 96-well falcon microtiter plate and measured at 585 nm. In total, three technical replicates were measured to estimate the pipetting and measuring error (figure 3).
Figure 3
96-well falcon microtiter plate containing the three technical replicates of the dilution series. Click to enlarge
First a broad concentration range was used to estimate the linear part. This range was made by a two-fold dilution series. When measuring the absorbance, LB medium was used as blank. Later the absorbance values of the blank were subtracted from the absorbance values of the standards. Then these values were divided by the absorbance values at 600 nm measured in the microtiterplate which gives an indication of the cell number. Eventually the values were corrected by setting the point with a concentration of 0 mM OHHL in the origin. These values were plotted in figure 4. The concentrations 2.56 mM and 5.12 mM were left out because these values were not distinguishable from the blank. This can be explained because the OHHL is dissolved in DMSO which lowers the growth of C. violaceum CV026. Between the concentrations 0.64 mM and 1.28 mM, the curve is stagnating. This is probably due to saturation of the medium or the inhibitory effect of DMSO. In a next step, a more narrow range was investigated.
Figure 4
First estimation of the OHHL standard curve. click to enlarge
In the second experiment, a range between 0.01 and 0.10 mM was investigated. The difference with the previous experiment is that the OD was first measured in 1 cm cuvettes and that the violacein was afterwards isolated. In this way, the plate reader does not contain cells. This optimisation is done, because we noticed in the previous experiment that violacein was produced more quickly in a culture tube than in a microtiterplate, probably due to the amount of supplied oxygen. Another reason for this working method is because estimating the amount of cells is more standardised by using cuvettes of 1 cm than using a microtiterplate.
Table 1 contains the processed values of the absorbance measurements. The processing is similar to the previous experiment. First the absorbance of DMSO was subtracted from the absorbance of the standards. Thereafter, these values were divided by the OD measured in 1 cm cuvettes. And finally, these values were corrected by putting the standard with a concentration of 0 mM OHHL in the origin.
Table 1: Absorbance at 585 nm divided by the OD
| Concentration (mM) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.10 | 0.09 | 0.08 | 0.07 | 0.06 | 0.05 | 0.04 | 0.03 | 0.02 | 0.01 | 0.00 | |
| T1 (corrected) | 0.126 | 0.072 | 0.110 | 0.093 | 0.057 | 0.083 | 0.052 | 0.049 | 0.076 | 0.011 | 0.000 |
| T2 (corrected) | 0.130 | 0.073 | 0.110 | 0.076 | 0.057 | 0.085 | 0.048 | 0.043 | 0.076 | 0.008 | 0.000 |
| T3 (corrected) | 0.121 | 0.071 | 0.078 | 0.091 | 0.054 | 0.083 | 0.053 | 0.049 | 0.051 | 0.008 | 0.000 |
| Average | 0.125 | 0.072 | 0.099 | 0.087 | 0.056 | 0.084 | 0.051 | 0.047 | 0.068 | 0.009 | 0.000 |
| Standard deviation | 0.004 | 0.001 | 0.019 | 0.009 | 0.002 | 0.001 | 0.003 | 0.003 | 0.015 | 0.002 | 0.000 |
In figure 5, our standard curve is plotted. A linear correlation between the absorbance and the concentration OHHL can be found. The variance of the technical replicates, visualised by the error bars, and the variance of the regression curve, shown by the R^2 value, can be explained by pipetting and measuring errors. Also, working with biological cells generates a background noise. This standard curve could give an estimation of bacterial AHL production. But it is important to keep in mind that there is background noise. Optimisation of this curve can be done by making more biological and technical replicas.
Figure 5
Standard curve ranging from 0 to 0.1 mM. The error bars represent the standard deviation between the technical replicates.
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be
