Team:SDSZ China/Experi-luci
Team:SDSZ China
Considering the possible drawbacks of the current detection methods, we decided to construct a plasmid that expresses firefly luciferase (Photinus Pyralis) to be utilized in bacteriophage detection.
A. Luciferase
Luciferase is the joint name for all enzymes in nature that induce bioluminescence. One of the most typical types is firefly luciferase from North American firefly (Photinus Pyralis). In corresponding reactions, the emittance of luminescence comes from the oxidation of luciferin, and most of the times the reaction system includes ATP and Mg2+.
Reaction process:
—Luciferase + ATP—> luciferyl adenylate + PPi
—Luciferyl adenylate + O2—> Oxyluciferin + AMP+ light
Considering that bioluminescence would be better detected and observed in seriously polluted water than simply chromogenic reactions, our team decided to construct plasmids with firefly luciferase sequence that can be induced in E.coli. cells to express luciferase proteins for further detection of phages in fecal-polluted water samples.
B. Overall Reaction Flow
As the cultured solution of strain BL21 of E.coli. cells is induced by IPTG and protein expression is confirmed by SDS-PAGE, different concentrations of bacteriophage are added to the same batch of bacteria solution.
-Bacteriophages of different concentrations attack E.coli.cell membranes to allow for the release of the expressed proteins inside the cells.
-Proteins of firefly luciferase leak into the background culture to interact with the substrates (Mg2+, ATP, D-luciferin).
-Enzymatic reaction occurs and bioluminescence is observed.
C. Plasmid Construction
1. Vector
At first we chose plasmid pET 28a（+） as vector for it contains T7 promotor and terminator as well as lac operators to initiate and control the expression of genes, plus pET plasmids are widely used in plasmid construction in our team’s projects in previous years.
However, we have found out that the pET 28 plasmids in our lab are somehow low in efficiency for plasmid construction. Thus, considering that pET 30a（+）plasmids contain nearly the same multiple cutting sites as pET 28a（+）, we adopted pET 30a（+）as our vector in the end.
2. Gaining sequence
The sequence of luciferase was gained by PCR from luciferase vector addgene-plasmid-37851-sequence-180888, with total length of 1653 base pairs.
primer sequence:
PCR product purification:
We adopted the traditional method of DNA purification instead of using a kit to gain a higher concentration of gene segment.
Protocol:
-Adding 0.1 volume sodium acetate (3M, pH=5.5) of the DNA sample to the tube, invert tube.
-Adding 2-3 volume ethanol (95%-100%) of DNA sample to the tube, invert tube.
-Precipitate 30minutes in -20 degrees Celsius.
-14000rpm centrifugation for 10-15 minutes, 4 degrees Celsius.
-Discard supernatant, add 500ul of ethanol（70%, kept in -20 degrees Celsius 20 minutes in advance）.
-14000rpm centrifugation for 2 minutes, 4 degrees Celsius.
-Discard supernatant, dry for 5 minutes.
-Suspend with 20-40ul of ddH2O for preservation.
3. Endonuclease Digestion and Ligation
While we were amplifying the gene segment of luciferase, blank plasmids of pET 30a(+) were transformed into clone strain Dh5a of E.coli.. Abundantly replicated plasmids were extracted， purified and concentrated to be prepared for digestion.
Nde1 and Nco1 are two unique enzyme cutting sites on pET 30a(+) plasmid. With the insertion of luciferase sequence, 6xHis tag, S-Tag and enterokinase site of the plasmid are deleted.
4. Transformation to clone strain
We have adopted two methods of transformation, one with liquid nitrogen and one with ice-bath. When transforming plasmids in Dh5a cells for cloning, ice-bath turned out to be a more efficient trial. Colony PCR’s results helped us select successful clones to culture, extract plasmids and transform into E.coli. strain BL21 for expression.
5. Plasmid DNA extraction and purification
After selecting corresponding successful clones to culture in liquid LB medium in a 10-30 ml system overnight, amplified plasmid DNA are extracted and purified. The purification protocol of plasmid DNA is the same as the purification of PCR products, yet the efficiency turned out to be lower.
6. Transformation to expression strain
Transformation to BL21 strain of E.coli. for protein expression was conducted through ice-bath according to standard protocol and clones were confirmed through colony PCR.
D. Protein Expression
Since plasmid pET30a contains lac operator sequence, IPTG induction was adopted to induce protein expression.
Before induction, cells were cultured in liquid LB medium 2ml for 2-3hours. OD600 was measured and IPTG induction began when OD600 was about0.4-0.6. OD600 of the culture was measured once in an hour since induction, and when the figure reaches about1.0, induction is stopped.
We ran SDS-PAGE to measure the protein expression status and confirmed the expression of firefly luciferase.
To further stabilize protein expression level ,we have considered adopting quorum sensing system in our design, but due to limit of resources the plan was not able to be realized.
E. Bacteriophage Introduction
1. Culture Medium
According to corresponding journals, we cultured our tailored E.coli. cells in MSB culture and added ATP, D-luciferin and Mg2+ ions to the medium serving as substrates for the enzymatic reaction.
2. Concentration Gradient of coliphages
We diluted the obtained coliphage solution into 1x, 2x, 5x and 10x solutions to test the intensity of bioluminescence of the reaction.
Since the lysis of E.coli. cells is not specifically restricted by the type of coliphages, we selected three types of coliphage in total for the reaction, which are strain SIYS and 242YS.
3. Reaction and data collection
The whole reaction system is confined in 4ml centrifuge tubes, and after phage solution was added to the E.coli. culture medium, the tubes were put into our darkroom so that luminescence can be better observed.
Photos were taken then to compare the luminance of each tube with different phage concentrations, and ideally a calorimetric card will be made for the following field test of the same batch of solution.
F. Results and Discussion
While introducing bacteriophages to the reaction system, we have adopted several ways to ensure the accuracy of our result.
To eliminate false positive results, we have purified phage solution through PES membrane to control the substance that causes cell lysis. Since the self-lysis cycle of E.coli. cells is about seven hours, we strictly control the total reaction time range within seven hours to prevent unwanted cell lysis.
To eliminate false negative response, we made sure to conduct negative control groups that contain the same circumstances except bacteriophages.
PSP SYSTEM
-Psp System Introduction
Psp system stands for phage shock protein system, including a set of intracellular and membrane proteins that are active in perturbation of cell membranes.
-Possible Design
We have considered applying Psp system to our design, inserting the sequence of luciferase or PPO on the downstream sequence activated by the binding of PspF protein. Under this circumstance, the expression of the protein is directly related to the attack of bacteriophages that perturbs cell membrane, and may possibly reduce false positive responses due to unexpected cell lysis other than caused by phage attack.
-Current problems and expected solutions
Since the complete sequence of promotor sigma 54 that induces downstream expression of PspA, B, C, D and E is not currently available and the required E.coli. strain was not obtainable by our team, we were not able to construct a plasmid accordingly.
However, we consider the idea of utilizing Psp system to control protein expression related to phage attack highly potential in future researches, and hopefully can explore more on this system after iGEM.
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