Team:SMS Shenzhen/Contribution
Contribute 1: Experiment for sacB Promoter Test
1. Experimental Report
1.1.The sacB gene and sacB promoter
The sacB gene usually extracted from bacillus subtilis encodes the secreted enzyme levansucrase (sucrose: 2,6-b-D-fructan 6-b-D-fructosyltransferase). The enzyme catalyzes hydrolysis of sucrose and synthesis of levans, which are high-molecular-weight fructose polymers. In E. coli, levansucrase activity is mostly located in the periplasm. The molecular basis of the toxicity is still unclear, but the toxicity could be due to an accumulation of levans which might encumber the periplasm because of their high molecular weight or a transfer of fructose residues to inappropriate acceptor molecules, which could thereafter have toxic effects on the bacterial cells. In our project, we make use of the sacB gene to kill the rest of E.coli that don’t play an effective role in coccid on leaves and scatter into the surroundings.
The sacB gene has been used in iGEM, but we didn’t find much description of sacB gene. Therefore, the efficiency of the promoter of sacB gene, which is a constitutive promoter, is uncleared. This experiment tests the efficiency of the promoter of sacB gene. We designed a plasmid with the promoter of sacB, ribosome binding side J61100, GFP and terminator B0015 on the backbone pSB1c3.
Figure 1 sacB promoter(534bp)+RBS（BBa_J61100）+green fluorescence protein +double terminator（BBa_B0015）+backbone（pSB1c3）
Figure 2 Sequence of sacB promoter
1.2. Vector Construction
We sent the plasmid to the Genscript company and had it man-made synthesized. To make a measurement next, we get a standard promoter BBa_J23100 into the identical plasmid backbone.
Figure 3 The standard promoter BBa_J23100 into the identical plasmid backbone
1.3. sacB Promoter Measurement
1.3.1 Abstract
In order to measure the strength of sacB promoter, standard measurement approaches for characterizing the sacB promoter were used so that it will be easier to be re-used by future teams.
We choose BBa_J23100(figure 4) from the Relative Promoter Units (RPUs) as the comparison to the sacB promoter. Three groups were set including an expertimental group(transformed with the sample), a positive control group(transformed with the standard J23100) and a blank controller. In each experiment there were also two parallel tests. After linking up the standard promoter BBa_J23100 into the identical plasmid backbone, we attained the OD600 to estimate the concentration of bacteria suspension with our linear function of detecting concentration of bacteria suspension in experiment 1. With the data of OD600,we controlled the bacteria in logarithmic phase. Then we made a measurement of the fluorescene of each groups.Using the fluorescein fluorescence standard curve made by A1~12 in 96 well plates and three groups of fluorescein intensity data,fluorescenece concentration was computed and histogram of two promoters was made to figure out the intensity difference from fluorescenece concentration.
1.3.2 Method summary and experimental data
1.3.2.1 Measurement of OD600
Using the spectrophotometer to get the estimated value of optical density of the 3groups, we made a conversion to the concentration of the bacterial suspension as an alternative independent variable of the expression intensity with the linear function of detecting concentration of bacteria suspension in experiment 1Y(Figure 5). Table 1 shows the Bacterial suspension Concentration[(CFU)/g] converted from the optical density[Abs].
1.3.2.2 Fluorescent Measurement
On the basis of data of OD600, we prepared the set-up of the ELISSA and calibration of fluorescence with each of the triplicates of the 6 groups, which was measured for its tripartite fluorescein. We also designed a set of precise concentration gradient to map the fluorescein fluorescence standard curve, after which the fitting function curve was attained.
For triplicates in each parallel group, the geometric mean of fluorescein [ABS] was calculated, and after all the geometric mean of each of the two parallel groups were attained. According to the arithmetic in terms of units, we divided it by the OD600 also averaged from each of the parallel groups.
To detecting inaccuracy in our measurement, we also saved the original database and use the corresponding data of OD600 to make enumeration in the same arithmetic, with which we have error bar in our contrastive histogram of the sacB promoter and BBa_J23100. Table 2 shows the original data attained from plate reader and the computation in terms of per unit in CFU. Table 3 shows the final result of the difference in the expressive quantity of GFP in these two promoters.
Table 2
1.3.2.3 Discussion
Using our original data, we made an analysis of the significant difference between the sacB promoter and the positive control BBa_J23100. It turned out that the P value of the data of the three groups each in pairs is far less than 0.05. We drew a conclusion that the data differs significantly. As a result, the promoter intensity of J23100>the sacB promoter and the expression of the sacB promoter in our engineering E.coli has reached an experimental verification.
2.Note
2.1Protocol
2.1.1 Transforming, Inoculation and Culturing
Day 1 A.M.
Preparation of Medium for Transformation
1. Solid medium 50mL
a) Get a clean beaker flask and make a mark on it
b) Measure Yeast extract 0.25g, Casein Tryptone 0.5g, NaCl 0.25-0.5g, and Agar 0.75g, using electronic balance
c) Pour them into the beaker flask
d) Add water till 50mL
e) Repeat the steps above and configure three flasks of solid medium
f) Pack them up
2. Liquid medium 100mL
a) Get a clean beaker flask and make a mark on it
b) Measure Yeast extract 0.5g, Casein Tryptone 1g, and NaCl 0.5-1g, using electronic balance
c) Pour them into the beaker flask
d) Add water till 100mL
e) Repeat the steps above and configure two flasks of liquid medium
f) Pack them up — 10:15
Sterilization
1. Get 5 culture mediums, 6 test tubes, and pipette tips
2. Put them into the sterilizer - 13:15
(Attention!! Only open the sterilizer when its temperature is lower than 90℃)
Day 1 P.M.
Transformation and Inoculation
1. Put competent cells in the ice box (a foam box filled with ice), keep it static and let solution form. This process takes about 5min.
2. Put the cells in 2 pre-cooled tubes separately, and each has 100μL. (One for positive control and another one for negative control.)
3. Add 2.5μL plasmids (2017 version, plane 4, hole 18A) to the positive control tube. Then mix the solution evenly and slightly. The solution has the resistance to CHL antibiotics.
4. Add 2μL dd water into the negative control tube (to control variables).
5. Place the mixture of plasmids and competent cells in the ice box for about 30 minutes.
6. Heat shock: take out the tubes and heat them for 30-90s in a water bath. Then, set them back in the ice box for another 5 minutes.
7. Add 800μL liquid medium without any antibiotics into the tubes. Set the tubes in a shaker in 37℃ to awake the bacteria. The process lasts for 30 minutes.
8. During the 30 minutes, a spreader as well as plate culture mediums with CHL and KANA antibiotics can be prepared. Prepare a plate without any antibiotics as blank control.
9. Take out the tubes and put them in the centrifuge machine under 5,000rpm for 3min.
10. Remove 800μL supernatant in both tubes, only leaving about 100μL to provide the liquid solvent for bacteria to float again.
11. Add 50μL mixture of liquid medium and bacteria from each tube onto the plates. Remember to tab the date, strain, and resistance on each plate
12. After the inoculation, place those petri dishes upside down in the thermostat box in 37℃. — 14:27
Day 2 A.M.
Shake-flask cultivation
Preparation: Put the following equipment into the clean bench prior to the experiment:
1) 7 Test tubes
2) 1 Alcohol lamp
3) 3 Petri dishes from yesterday
4) Pipettes (different capacities)
5) Pipette tips (difference sizes)
6) 2 bottles of LB medium with sucrose
7) 1 Marker
Experimental procedures:
1. Turn on the UV lamp in the clean bench for 30 minutes.
2. Turn on the fan in the clean bench for 5 minutes.
3. Wear gloves, sanitize them with alcohol.
4. Light the alcohol lamp.
(Note: the following procedures 5-9 need to be conducted within 10 cm of the flame of the alcohol lamp.)
5. Take 7 test tubes, mark each with markers
6. Burn the top of the test tube, and fill in 10 mL of LB medium. (Repeat this procedure for 6 times)
7. Add antibiotic into the test tubes. For the blank control group, add the same amount of distilled water.
8. Use pipette tips to pick up the bacteria on the peri dishes from yesterday, and put it into the 7 test tubes, and remember to also put the tips into the test tubes. (For each kind of bacteria, use 2 test tubes.)
9. Add 10 mL of LB medium into the last test tube.
10. Put all the test tubes into the incubator, and set it at 17 ˚C for 16 hours.
Day 3 P.M.
Measurement of Optical Density:
1. Take one each of the bacteria from yesterday, sanitize the outer glass of the test tubes, and put it into the clean bench.
2. Take a cuvette (upside down), sanitize it with alcohol, and put it into the clean bench.
3. Clearly mark the cuvette, add in the corresponding bacteria, and the blank control group.
4. Determine the optical densities.
Drip in each well for 100 µL
A for the 1st sample
B for the 2nd sample
C for the 3rd sample
D for the 4th sample
Settings for the microplate reader:
Excitation 485nm
Emission 530/30 (or close to this as possible)
Contribute 2: Help Characterize the Structure of Bacillus Bubtilis Levansucrase
In order to help characterize the structure of bacillus subtilis levansucrase in Parts, SMS_Shenzhen uploaded the 3D structure of this enzyme.
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