Team:FAFU-CHINA/Design
Background
Apiculture in the world
No one knows since when did humans begin to attempt to domesticate honeybees, just like no one knows exactly how important these little creatures can be to human civilization and world environment.
Honeybees may be most famous for the stored honey they serve up, but there are a few more useful things they do as well. They produce beeswax, which gives us the manufactures of candles and many other sorts of products. They produce royal jelly, which is extremely famous for preserving people’s health. Moreover, they pollen plants, which is the most fundamental ecological process and makes everything in nature possible.
Today, in China, apiculture is a vital economic generator for the country, producing average 245,000 metric tons and estimating at 238,000 metric tons of consumption in honey per year. In America, it creates 210,000 direct jobs for people, and it is a crucial element of U.S agriculture, which relies on apiculture for pollination. it is impossible to define the economic value of apiculture.
According to a data released by Times, almost 1/3 of food of the world is dependent on honeybee’s pollination. If all the honeybees die out, thousands of plants will follow, which could leads to millions of people starving in the following years.
Einstein once said that’if all the bees die out, humans will follow a few years later.’ It is unsettling to inform you that, honeybees have started to disappear. In recent years，beekeepers have witnessed an annual 30% losses in their colonies. For the Europe alone, bees are greatly declining, from 5 million hives in 1988 to 2.5 million today. Since 2006, a phenomenon called Colony Collapse Disorder has spread to every corner of the world. We don’t exactly know what is causing it. The only thing we know is that it is pretty serious, and measures must be taken immediately.
Sacbrood Virus
Sacbrood Virus has been a scourge to apiculture over the world for almost half of a century. Sacbrood Virus is a typical RNA virus. It causes failure of larval pupation, and results in larval death. With SBV accumulating beneath larval skin, Infected larva changes in color from pearly white to pale yellow, forming a brown gondola-shaped scale after death. Different Sacbrood virus samples originate from various geographic regions. In China, it is classified as Chinese Sacbrood Virus (CSBV). According to a research from Jilin University, in some provinces of China, 60 percent of bee tribes carries CSBV, and the death rate of larvae is almost 100 percent during outbreaks.
CSBV is one of the main causes of the significant decrease of Chinese honeybee populations in the past 40 years. So far, no effective control method exists for this bee disease, besides the prevention strategy, the only one beekeepers adopting currently. FAFU-China team aims to investigate the possibility of controlling this disease through silence of CSBV’s RdRp (RNA-dependent RNA polymerase) gene by using RNA-interference technology.
RNA-interference
The RNA-interference naturally exists in many eukaryotes, including honeybees. It plays an crucial role in defending somatic cells against viruses. The biological process is known for it’s ability to inhibit gene expression by causing the destruction or silence of specific messenger-RNA molecule.
Double-stranded ribonucleic acid (dsRNA) molecules is essential to RNA-interference. In the very beginning, the Dicer enzyme initiates the biological process by cleaving dsRNA into small interfering RNA (siRNA). Next, the small interfering RNAs are soon separated into two single strands. The positive-strand which is also known as guide-RNA is then integrated into an active RNA-induced silencing complex, RISC. RISC can bind to it’s target mRNA by base-pairing and cleave it, preventing the mRNA from producing a protein.
Theory
Replication of CSBV
As a typical RNA virus, CSBV’s replication process is initiated once the messenger RNAs of CSBV, which is CSBV’s original genetic information, are injected into somatic cells. However, a essential complex for the replication of genetic information of CSBV is lacked in the somatic cells of honeybees, RNA dependent RNA polymerase, RdRp in short. RdRp can be synthesized directly by ribosome of honeybees using mRNA of RdRp as temperate. With the existence of RdRp, the replication of genetic information of CSBV can be completed and CSBV will soon spread out infect the whole organism.
This year，FAFU-China aims to investigate the possibility of controlling this disease by inhibiting the expression of RdRp gene through RNA-interference.
CSBV.Silencer 1.0
We designed a engineered bacteria capable of producing double-stranded RNA of RNA dependent RNA polymerase （RdRp）to initiate the process of RNA-interference. We inserted the RdRp gene into plasmid L4440, and inserted T7 promoters in the upstreams of both strands of the plasmid. two contemporary messenger-RNAs of RdRp can be synthesized simultaneously after T7 polymerases detect the promoters in both strands and start the transcription from 5 prime to 3 prime.
The contemporary messenger RNAs will then bind together into double stranded RNA by base-pairing. Long dsRNA of RdRp is then cleaved by Dicer enzyme and short small interfering RNA is formed. small interfering RNA will soon separate into two single strands. One is called passenger RNA, which is soon degraded. And the other strand called guide RNA will then integrated into an RNA Induced Silencing Complex, RISC in short. RISC then binds to the specific sequence of mRNA of RdRp and destruct it, thereby preventing it from being used as translation template to produce RdRp.
We transferred plasmid L4440 into E.coli so that dsRNA of RdRp can be largely produced. Then we mixed our product, CSBV.Silencer, into forage of honeybees, and feed it to infected hives. After the infected honeybees take in CSBV.Silencer, the replication process of CSBV in will be destructed in the somatic cells of honeybees, for the essential RdRp complex can not be formed.
CSBV.Silencer 2.0
On the basis of CSBV.Silencer 1.0, we decided to improve our product,out of two concerns.
In the field of food industry, safety concern is always a sensitive problem. E.coli is always taken as potential pathogen by consumers unconsciously，and because of that, we decided to replace E.coli by Yeast which originally exists in honey.
If we want to transfer CSBV.Silencer into honeybees, we need to consider the influence imposed by immune system and digest system. We need to find a way to prevent our product from being degraded by immune cells and digestive solution. The need to enable the engineered bacteria getting through the digest system without being harmed also requires us to replace the E.coli by using eukaryote as carrier.
However, if we want to use eukaryotic system to produce the dsRdRp, there is still a problem needed to be solved. In eukaryotic system, there is no T7 polymerase, which causes failure in transcription of L4440, leading dsRdRp unable to be synthesized. To solve this, we have another plasmid capable of producing T7 polymerase, PyES2.
After galactose induces Gal1 promoter, activating RNA polymerase to transcript the gene of T7 RNA polymerase, mRNA of T7 RNA polymerase can be expressed and T7 polymerase is produced. the produced T7 polymerase can go back to the T7 promoter in the upstream, and continue making more T7 polymerases. The circulation of making T7 polymerase ensures the abundance of T7 polymerases, which actually accelerates the process of producing dsRdRp.
Our next product, CSBV·Silencer 2.0, will be the engineered yeast containing plasmid PyES2 and plasmid L4440. However, in this stage, we have only come up with the idea and are still trying to recombine the plasmid PyES2. As long as we have the recombined plasmid PyES2 ready, we will then transfer PyES2 and L4440 into yeast, and have our product applied to practice.
Meanwhile,we noticed that IPTG could induce the expression of dsRdRp in HT115, and galactose could improve the efficiency of GAL1 in eukaryotic system
Easier to spread
Eusocial insects like ants, honeybees, bumble, termites have the habit of cooperative care of young. The larvae in the family are unable to forage and feed themselves, and because of that, the food delivered by workers are essential. this habit enables CSBV.Silencer flow through the whole insect family rapidly.Through the mechanism, CSBV.Silencer can be diffused easily and the effect can be detected rapidly.
Similarly, the mechanism can be adopted in the prevention of other virus disease of eusocial insects or biological control. We are planning to develop a medicine of termite control through silence of certain gene. We predict that the medicine can be delivered with food throughout the whole colony, and achieve the purpose of pest control.
Protocol
Whole RNA Extraction
We use Trizol to extract the whole RNA of CSBV,and the detail protocol is showed as bellow:
1.Take 50-100mg tissue stored in -80℃ or liquid nitrogen. Grind the tissue into powder in mortar with liquid nitrogen. Transfer the powder into a 1.5ml EP tube(RNase-Free).
2.Add 500μl Trizol, vortex to mix thoroughly and let the tube sit at room temperature for 10 min.
3.Centrifuge at 12,000 x g for 10 min at 4℃.Transfer the supernatant into a new 1.5ml EP tube(RNase-Free).
4.Add 100μl chloroform, vigorously shake with hand for 15s and then let the tube sit at room temperature for 10 min.
5.Centrifuge at 12,000 x g for 15 min at 4℃. Transfer the upper colourless aqueous phase into a new 1.5ml EP tube(RNase-Free).
6.Add 250μl isopropanol, turn the tube upside down many times for around 10 min, and let it sit at room temperature for 10 min until the white floccule arise,and then centrifuge at 12,000 x g for 10 min at 4℃.
7.Discard the supernatant,add 500μl,75%ethanol with DEPC,and mix thoroughly.Centrifuge at 7,500 x g for 5 min at 4℃ and discard the supernatant.
8.Dry it at room temperature or in the bechtop for 15 min.
9.Add 15-20μl RNase-Free water to dissolve RNA when the precipitate become transparent.
10.Store RNA at -80℃.
RT-PCR and PCR
We amplify DNA of CSBV by RT-PCR and PCR. And the detail systems and programs are showed as bellow:
1.RT-PCR（using M-MLV Reserve Transcriptase kit bought from Promega company
Component   ; Volume(μl)
Whole RNA of CSBV 4.0
dNTP Mixture(10mM) 4.0
Reserve primer 2.0
70℃ pre-denaturation 10min
Component(add in order) Volume(μl)
5×M-MuLV buffer 4.0
RNase free water 4.5
M-MLV Reserve Transcriptase 1.0
42℃,1h;72℃,15min
Store cDNA at -20℃
2.PCR
（1）System(50μl)
10×PCR Buffer 5.0μl
2.5mM dNTP Mix 2.0μl
Template(cDNA) 2.0μl
Primer F 2.0μl
Primer R 2.0μl
Dream Taq DNA pol 0.5μl
ddH2O 36.5μl
Volume 50.0μl
(2) Program
94℃ 5min
94℃ 30s
55℃(Tm-5℃) 30s
72℃ T(1min/kb) 2 34
72℃ 10min
16℃ ∞
qPCR
We use GoTaq® qPCR Master Mix Kit(buy from Promega company) and Eppendrof Mastercycler® ep realplex Real-Time PCR to do qPCR.
(1)System(20μl)
SYBR Premix Ex TaqTM 2×mix 10.0μl
Template(cDNA) 1.0μl
Primer F 0.4μl
Primer R 0.4μl
RNase-Free water 8.2μl
Volume 20.0μl
(2) Program
95℃ 2min
95℃ 20s
60℃ 20s
72℃ T(1min/kb) 2 39
72℃ 10min
16℃ ∞
Plasmid Extraction
We use E.Z.N.A.® Plasmid DNA Mini Kit I bought from OMEGA bio-tek company to
extract plasmids and the detail protocol is showed as bellow:
1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at
37°C with vigorous shaking (~ 300 rpm).
2. Centrifuge at 10,000 x g for 1 minute at room temperature
.
3. Decant or aspirate and discard the culture media.
4. Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.
5. Transfer suspension into a new 1.5 mL microcentrifuge tube.
6. Add 250 µL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.(Avoid vigorous mixing)
7. Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.
8. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
9. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
10. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column.
11. Centrifuge at maximum speed for 1 minute.
12. Discard the filtrate and reuse the collection tube.
13. Add 500 µL HB Buffer.
14. Centrifuge at maximum speed for 1 minute.
15. Discard the filtrate and reuse collection tube.
16. Add 700 µL DNA Wash Buffer.
17. Centrifuge at maximum speed for 1 minute
.
18. Discard the filtrate and reuse the collection tube.
19. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
20. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
21. Add 30-100 μl Elution Buffer or sterile deionized water directly to the center of the column membrane.
22. Let sit at room temperature for 1 minute.
23. Centrifuge at maximum speed for 1 minute.
24.Store DNA at -20°
Gel Extraction
We use E.Z.N.A.® Gel Extraction Kit bought from OMEGA bio-tek company to recover our target fragments after AGE and the detail protocol is showed as bellow:
1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.
2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.
4. Add 1 volume Binding Buffer (XP2).
5. Incubate at 60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
6. Insert a HiBind® DNA Mini Column in a 2 mL Collection Tube.
7. Add no more than 700μl DNA/agarose solution from Step 5 to the HiBind® DNA Mini Column.
8. Centrifuge at 10,000 x g for 1 minute at room temperature.
9. Discard the filtrate and reuse collection tube.
10. Repeat Steps 7-9 until all of the sample has been transferred to the column.
11. Add 300 µL Binding Buffer (XP2).
12. Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
13. Discard the filtrate and reuse collection tube.
14. Add 700μl SPW Wash Buffer.
15. Centrifuge at maximum speed for 1 minute at room temperature.
16. Discard the filtrate and reuse collection tube.
17. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
18. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
19. Add 30-50μl Elution Buffer or deionized water directly to the center of the column membrane.
20. Let sit at room temperature for 2 minutes.
21. Centrifuge at maximum speed for 1 minute.
22.Store DNA at -20°C.
CTAB Extraction of dsRNA
1.Pipet 2mL bacterium suspension, centrifuge at 5,000rpm for 1 minute.
2.Discard the supernatant, add 500μl TE Buffer(10mmol· L-1Tris, 1mmol· L-1EDTA,PH7.5) and resuspend.
3.Add 30μl 10%SDS,mix thoroughly and put it in 37℃ incubator for 1h.
4.Add 100μl NaCl(5mol· L-1), mix thoroughly.
5.Add 80μl CTAB/NaCl, mix thoroughly, 65℃ water bath for 10min.
6.Add isometric phenol/ chloroform/isoamylol mixture(25:24:1), mix thoroughly and centrifuge at 12,000rpm for 5 minute at 4℃.
7.Transfer the supernatant into a new 1.5 mL microcentrifuge tube. Add 3/5 volume of it isopropanol. Centrifuge at 12,000rpm for 10 minute at 4℃.
8.Discard the supernatant, add 500μl 75% ethanol, vortex to mix thoroughly, and centrifuge at 6,000rpm for 5 minute at 4℃.
9.Vacuum dry for 10minute, and digest with DNase(1U), RNase(1U) respectively.
10.Store dsRNA at -80℃ or use for next experiment immediately.
Dual-enzyme digestion
1、
Vector 10.0μl
Restriction enzyme(EcoR1,Pst1) 0.5μl
Buffer(10×H) 2.0μl
ddH2O 7.0μl
Volume 20.0μl
37℃ metal bath,8h or overnight.
2、AGE, recover target fragments by gel extraction.
Linkage and transformation
1.Linkage system(10μl): Target fragment 4.0μl, Vector 1.0μl, 5×Buffer 2.0μl, T4 ligase 1.0μl, ddH2O 2.0μl. 4℃, overnight.
2.Add the product of linkage into 50μl DH5αcompetent cell, pipet up and down gently to mix thoroughly, and put it on ice for 30min.
3.Heat in 42℃ water bath for 90s, and take it out immediately and put it on ice for at least 2min.
4.Add 200μl LB medium without any selective antibiotic. Incubate for 45min at 37℃with vigorous shaking (~ 220 rpm).
5.Pipet 150μl bacterium suspension and smear it on LB plate with the appropriate selective antibiotic. Culture in 37℃ incubator,overnight.
CSBV.Silencer 2.0
Preliminatry experiment
The effect of different homologous dsRNA of CSBV to the expression of CSBV’s mRNA.
To find the most effective homologous dsRNA of CSBV for our project, we tested the pupation rate of honeybee larvae under different dsRNA. We added different homologous dsRNA of CSBV (dsHelicase, dsProtease, dsRdRp and dsVP1) into forage and fed to the infected larvae. After 12 hours, We fed the larvae with extracted fluid of CSBV, and kept feeding it in the following day. According to the statistics we collected, infected larvae fed with dsRdRp have the highest pupation rate among all the experimental groups.
The effect of CSBV.Silencer in curing the disease
We fed the infected hives with our CSBV·Silencer, and collected the following statistics under the help of professional beekeepers from College of Bee Science.
1.The decrease of the number of infected larvae.
Compared with the control group, it is quite obvious that the infected larvae in medium concentration group and in high concentration group dropped dramatically by 91.44 percent and 94 87.91 percent after 14 days while the infected larvae in control group showed 65.82 percent increase after 7 days.
2.The effect of dsRdRp on the number of sealed brood.
The number of sealed brood is another indicator of the effect of CSBV. It is an necessary stage of larval pupation. In this graph, the number of sealed brood in medium concentration and high concentration increases by about 23.68% and 30.10% after 21days. Similarly, the result witnesses a significant improvement in the pupation rate.
3.The increasing population.
We counted the number adult honeybees to study the changes of population.The changes are just as you see. For the high concentration group alone, the growth of population had reached almost 60% in just 21 days. On the contrary, the control group showed a 27.78% decrease after 21 days.
These statistics directly proved that, our product, CSBV.Silencer 1.0 does have a remarkable effect in the treatment to Sacbrood disease.
The effect of galactose to induce GFP expression at different concentrations and different time./div>
1.The galactose at three different concentrations can induce GFP expression efficiently and have little influence on the level of GFP
We transformed GFP-pYES2 into the yeast and let the recombinant strain grow to mid-log phase in YEPD medium containing 2% glucose. And then we transferred it to medium, containing 2%, 0.1% or 0.02% galactose, and assayed for GFP fluorescence.
According to the statistics we collected, we find that the production of GFP protein is quite high even though at 0.02% galactose and with the increase of its concentration, the level of GFP have no remarkable change.
2.GFP production sharply increases and a peak shows up in about 24h under the induction of galactose.
The yeast transformants were grown in YEPD medium. At a cell density of 2 ×107 ml-1,
cultures were washed and galactose added to a final concentration of 2% and the fluorescence value of samples was measured every 5h.
As the diagram shown, we can find that GFP production sharply increases in 10h and a peak shows up in about 24h and then it decreases gradually.
In conclusion, we can find using 0.02% galactose to induce the expression of GFP in 24h is a reasonable and efficient way to get the most production of GFP protein. It means that we can induce the expression of T7 RNAP gene efficiently under the same condition.
CSBV.Silencer 2.0
CSBV.Silencer 2.0
CSBV.Silencer 2.0
CSBV.Silencer 2.0
CSBV.Silencer 2.0
CSBV.Silencer 2.0
CSBV.Silencer 2.0
Communication Design
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Communication Design
Communication Design
Communication Design