Team:XMU-China/Content5

1 Circuit Construction

1. Material

2. PCR Polymerase Chain Reaction (PCR)

3. Digestion

4. Ligation

5. Transformation

2 SDS-PAGE

1. Preparation

2. Bacteria culture

3. Protein electrophoresis

3 Microfluidic Protocol

1. Pre-procession of Dh5a

2. Pre-procession of LB

3. Wetting the chip & Capturing images

4 SDS-PAGE

1. Preparation

2. Bacteria culture

3. Protein electrophoresis

Material

1.1 Culture medium: LB (Luria-Bertani)

LB medium: 5g Yeast Extract, 10g Tryptone and 10g NaCl, add ddH2O to 1L.
LB solid medium: 5g Yeast Extract, 10g Tryptone, 10g NaCl and 15 g agar, add ddH2O to 1L.

1.2 Antibiotic

Antibiotic	Stock conc.	Final conc.	Solvent	Sterilization
Ampicillin	100 mg/ml	50 ug/ml	ddH2O	filtration
Chloramphenicol	25 mg/ml	25 ug/ml	Absolute ethyl alcohol
Tetracycline	40 mg/ml	20 ug/ml	75% absolute ethyl alcohol
Kanamycin	25 mg/ml	25 ug/ml	ddH2O	filtration

Stock in -20°C

1.3 Plasmid (Biobrick)

Part	Backbone	Type	Location	Size (bp)
BBa_K546000	pSB1C3	Signaling	2013-P1-12D	1964	2070
BBa_I763020	pSB1C3	Intermediate RBS-GFP-TT	2013-P3-11H	914	2070
BBa_F2621	pSB1A2	Signaling	2013-P2-21F	1158	2079
BBa_K546001	pSB1C3	Device	2013-P1-12F	2135	2070
Ba_J04450	pSB4K5	Reporter	2013-P5-5G	1429	3409
BBa_J04450	pSB3T5	Reporter	2013-P5-7C	1429	3241
BBa_J04450	pSB3T5	Reporter	2013-P3-3H	1429	3241

1.4 Strain

Strain	Application	Source
E. coli DH5a	cloning	Lab stock
E. coli BL21(DE3)		Lab stock
E. coli BL21		From Taiwan
E. coli MG1655

1.5 Primer

Part	Backbone	Type	Location	Size (bp)
				part	backbone
BBa_K546000	pSB1C3	Signaling	2013-P1-12D	1964	2070
BBa_I763020	pSB1C3	Intermediate RBS-GFP-TT	2013-P3-11H	914	2070
BBa_F2621	pSB1A2	Signaling	2013-P2-21F	1158	2079
BBa_K546001	pSB1C3	Device	2013-P1-12F	2135	2070
Ba_J04450	pSB4K5	Reporter	2013-P5-5G	1429	3409
BBa_J04450	pSB3T5	Reporter	2013-P5-7C	1429	3241
			2013-P3-3H

2. PCR Polymerase Chain Reaction (PCR)

2.1 General PCR

Reaction system:

Template1	1 μl
Forward Primer	2 μl
Reverse Primer	2 μl
dNTP Mixture	8 μl
10*Ex Buffer	5 μl
Ex Taq	0.5 μl
ddH2O	31.5 μl
Total Volume	50 μl

Reaction condition:

94 oC	4 min
94 oC	1 min	30 cycles
55 oC2	0.5 min
72 oC	1.5 min3
72 oC	10 min
4 oC	∝

2.2 Fusion PCR

Every biobrick has four sites: EcoR I, Xba I, Spe I and Pst I. When two parts were ligated after digestion by Xba I and Spe I, an 8 bp-size fragment (TACTAGAG) will appear between two parts. If we want to add a fragment in the end of a CDS, for example a fast-degradation tag LVA, the additional 8 bp-size fragment will affect the expression of the CDS. Fusion PCR will resolve this problem. Firstly, two pair primers are designed: F1 and R1 for gene A, F2 and R2 for gene B. The 5'-end of R1 and F2 have about 21-bp homologous region. Secondly, gene A and gene B are amplified via PCR independently. Thirdly, the purified gene A and gene B are fused together via PCR with F1 and R2.

2.3 Colony PCR

Reaction system:

Template1	colony
Forward Primer	1 μl
Reverse Primer	1 μl
dNTP Mixture	4 μl
10*PCR Buffer	2.5 μl
rTaq	0.25 μl
ddH2O	16.25 μl
Total Volume	25 μl

Reaction condition:

94 oC	4 min
94 oC	1 min	25 cycles
55 oC	0.5 min
72 oC	1.5 min
72 oC	10 min
4 oC	∝

3. Digestion

3.1 Digestion for confirmation

Reaction System:

Single Digestion		Double Digestion
Plasmid	5	Plasmid	5
EcoR I	1	EcoR I	1
/		Pst I	1
10*H buffer	1	10*M buffer	1
ddH2O	3	ddH2O	2
Total Volume	10	Total Volume	10
Total Volume

Reaction condition: 37°C for 1 hours.

3.2 Digestion for ligation

Reaction System:

A + B		B + A		Volume (μl)	Volume (μl)
A	B	A	B	30	50
EcoR I	EcoR I	Xba I	Spe I	3	5
Spe I	Xba I	Pst I	Pst I	3	5
10*H buffer	10*M buffer	10*M buffer	10*H buffer	4	8
ddH2O				0	12
Total Volume				40	80

4. Ligation

	Volume (μl)
Insert	V1
Vector	V2
10*ligation buffer	1
T4 Ligase	1
Total Volume	10

5. Transformation

5.1 Chemical Competent Cell Preparation (E.coli)

Activate the E.coli strain on LB-plate from glycerol stock under the condition of 37^oC for 12 hours.

Pre-culture single-colony in 10 mL LB medium under the condition of 37^oC, 200 rpm for 12 hours.

Add 200 μl pre-culture into 20 mL LB medium under the condition of 37^oC, 200 rpm for 2 hours.

Place on ice for 30 min. Aliquot into sterile 1.5 mL tubes and spin down at 4000 rpm for 10 min at 4 ^oC, then discard supernatant.

Gently re-suspend each pellet with 1 mL0.1 Mice cold CaCl₂-MgCl₂ solution. Centrifuge 4000 rpm for 10 min and discard supernatant. 

Re-suspend each pellet on ice in 200 μL 0.1 M ice cold CaCl₂-MgCl₂ solution

5.2 Chemical Competent Cell Preparation (E.coli with plasmid(s))

When you prepare the competent cell of E.coli with plasmid(s), you need add half-concentration of appropriate antibiotic(s) in LB.

5.3 General Transformation

Add 1 μL plasmid or 10 μL ligation system into 100 μL fresh chemical competent cells and mix gently. Leave on ice for 30 minutes.

Heat pulse tubes in 42^oC water bath for 90 seconds, then place them on ice for 10 minutes immediately.

Incubate for 1~2 hr with 200 rpm shaking at37°C. When the antibiotic of vector is ampicillin, the time of incubate is 1 hr.

Add 400 μL fresh LB medium into each tube.

Add 1 μL plasmid or 10 μL ligation system into 100 μL fresh chemical competent cells and mix gently. Leave on ice for 30 minutes.

Spread 100 μL on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37°C

Two or three plasmids Transformation

Usually we just transform one plasmid into host cell, but sometimes we need transform two or three plasmids. Plasmid compatibility is considerable. Generally different replication origin means they are compatibility. We transform plasmids one by one from the low copy number to high copy number.

Preparation

2.1 Bacterial strains:

The experimental strains:E.coli

2.2 The main instruments and reagents
Instruments

autopipets, electrophoresis meter, Vortex oscillator, Labscan Scanner, decolorization table, desk centrifuge, temperature metal bath, electronic balance, acidometer, enzyme-labeled instrument, superclean bench, constant temperature vibrator, shaking table.

Reagents

LB liquid medium: yeast powder 5.0 g ;peptone 10.0 g ;sodium chloride 10.0 g;deionized water to produce 1000 ml medium.

LB solid medium: Put 1.5 g agar into every 100 ml liquid medium.

Preparation of electrophoretic solution

ddH₂O;4 X Running Gel Buffer (PH 8.8,1.5 M Tris-HCl);4 X Running Gel Buffer (pH 6.8,1.5 M Tris-HCl);Monomer Solution;10% sodium dodecylsulphate(SDS);10% Ammonium Persulfate(APS);N,N,N',N'- tetramethylethylenediamine(TEMED);loading buffer;Tank Buffer;industrial alcohol; Marker ; dying liquor;destainer.

(1) Preparation of 4 X Running Gel Buffer (pH 8.8,1.5 M Tris-HCl): 36.3 g Tris-base is dissolved in 150 ml deionized water. Then use HCl solution to adjust the pH of solution to8.8. At last use deionized water to produce 200 ml solution.

(2) Preparation of 4 X Running Gel Buffer (pH 6.8,1.5 M Tris-HCl): 3.0 g  Tris-base is dissolved in 40 ml deionized water. Then use HCl solution to adjust the pH of solution to 6.8. At last use deionized water to produce 100 ml solution.

(3)Preparation of 10% SDS: 10g SDS is weighted before being dissolved . Use  distilled water to produce 100ml solution.

(4)Preparation of 10% APS: 10g APS is weighted before being dissolved. Use  distilled water to produce 10ml solution.

(5) Preparation of 1X Tank Buffer: 30.28 g Tris-base, 144.13 g glycine and 10 g SDS is weighed before being dissolved. Use distilled water to produce 1000ml solution. Use the solution as-prepared 100 ml mixed with 900 ml distilled water to dilute 10 times.

(6) Preparation of Kaumas Coomassie brilliant blue staining solution: 0.5 g Coomassie Brilliant blue R250 is weighted before being dissolved in 800 ml industrial alcohol. After dissolving, 140 ml acetic acid is added and Use  distilled water to produce 2000ml solution

(7) Preparation of destainer: 200 ml industrial alcohol is mixed with 50 ml acetic acid. Use distilled water to produce 1000ml solution.

Preparation of 10% gel.

2.2. Bacteria culture

Strain activation

Pick Escherichia coli colony from LB plate and subculture it into 20 ml conical beaker with 50 ml LB broth followed anaerobic culture 12 h under 37 °C . Then culture 1% concentration into conical beaker with 50 ml LB broth to subculture, which is the activated germ liquid.

Strain culture

Subculture 1% activated germ liquid into every conical beaker with LB broth. Then culture the microbe in the shaking table ,under 37 °C ，200 rpm.

2.3 Protein electrophoresis

Preparation of Samples

- The samples to be tested are cultured in the Basal Medium with appropriate antibiotics, and take 200 μL bacteria liquid to determine its OD₆₀₀ at appropriate time.
- Dilute or concentrate the next 200 μL bacteria liquid in order to let the OD₆₀₀ equals to 4.0 while the computational formula is the actual OD₆₀₀ * 200=2.0 * X, and X presents the total volume of the bacteria liquid after being diluted or concentrated while its unit is μL as well.
- Add 30 μL diluted or concentrated liquid into corresponding 1.5 mL centrifugal tubes, then mix up them with 10 μL loading buffer.
- Put these centrifugal tubes into metal bath and heat them in 100 °C in around 5 to 8 min, then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is what we need.

Manufacture Albumen Gel

- Prepare a clear centrifuge tube in the capacity of 50 mL, and make running gel, high concentration one, following the formula below. Then mix up them and pour the mixture into a glass pane.

- Add some absolute alcohol to planish the top of gel. There will be approximately 60 min for its solidification.

- After solidification, pour out the alcohol and make stacking gel following the formula below. Then mix them up, add the solution onto the running gel in the glass panes until it being filled up with the gel. Insert a clean comb into stacking gel. Wait for about 40 min for stacking gel solidification.

Electrophoresis

- Take out the glass pane with finished gel and then fasten it in an electrophoresis tank. Add some 1×Tank Buffer to detect whether liquid leak or not.     
- Take out the comb slowly and use pipette to add approximately 10 to 20 μL processed samples into the wells in stacking gel.
- Add 1×Tank Buffer until the liquid level is above the platinum line in the electrophoresis tank.
- Cover up the electrophoresis tank and connect it with the electrophoresis device. Set the program 120 V- 60 min and start it up.
- When the green marker band run to the bottom of running gel, stop the device.

Dyeing (Colloidal Coomassie Brilliant Blue)

- Take out the gel and put it into a clean petri dish. And add appropriate Commassie Blue Staining Solution. Please make sure that the solution can cover all the gel.
- Put the petri dish onto the orbital shaker and dye for approximate 1 h.
- Pour out the staining solution then add enough destaining solution. Destain about 30 min.
- Renew the destaining solution for about 2 or 3 times until the blue background of gel being taken off.
- Pour out the destaining solution and add appropriate water to clear it.

Scanning

Scan the processed gel and save the picture for analysis.

It’s a great honor for me to introduce our attractive “byproduct”——Bio Bang. We designed these special card for these high school students who were the top ten on our scoring list. These sets of cards——we called them Bio Bang——were inspired by War Within Three Kingdoms, which are very popular in China，just like Bang！ in America. It consists of abundant role-relationship and mutual promotion and restraint between every cards. The Bio Bang is themed with synthetic biology, including genetic engineering experiments, biological materials and equipment. We’ve also assimilate knowledge of biosafety into these set of cards in order to popularize knowledge of biosafety in lab, public and environment. This kind of propaganda tool is very useful to make different knowledge about genetic engineering and biology get across to high school students. Some example of them are shown as follows:

We’ve dreamed about perfecting the rules and designing and sold it out to raise money for our project. But it didn’t come true owing to our tight schedule. However, we decided to make a English version Bio Bang as presents for other iGEMers. Just wait for us coming!

Surveys on The Popularization of Synthetic Biology and iGEM in high school students of Fujian Province

Our team conducted a synthetic biology survey before our lecture entitled "Contemporary introduction to synthetic biology". hWe wanted to compare how much they knew about synthetic biology and iGEM before the lecture with what their knowledge about them after our presentation in order to assess our work. We’ve prepared 300 questionnaires and handed out 227 of them. Among these drew-back questionnaires, 180 of them are valid. We put 12 questions on the questionnaire as below.

1. Have you ever heard about synthetic biology before our lecture?
2. If you have, in which way it was accessible to you?
3. Which parts do you think are the most intriguing about synthetic biology after our lecture?
4. Have you ever heard about iGEM before our lecture?
5. If you have, in which way it was accessible to you?
6. Are you willing to participate in iGEM after our lecture?
7. If your answer is no, why?
8. What’s your reason for participation, why?
9. If you participate in iGEM in which aspects do to want to get supports?
10. How much time can you spend on iGEM?
11. If you participate, you and your family are willing to afford in which aspects? Some survey results are shown as below:

Discussion
Two major findings have been obtained from this analysis.

First of all, we drew a conclusion that almost everyone learned a lot from our lecture although they tended to be unfamiliar about the details of synthetic biology. Comparing the number of people who know about iGEM and the number of people who are willing to participate in iGEM we think the number of people who are interested in iGEM increases nearly 60%. Just look at the reasons why they don’t want to participate, most people chose no time and no supports. Only few people claimed that they were not interested in it and far less people said they didn’t like biology at all. Considering that our high school students are under so much pressure from National College Entrance Examination and some high school are too poor to afford technologies and apparatus, our results indicated a really good reality that almost everyone was willing to accept this new science and technology.

Second, as for how to popularize iGEM and synthetic biology, we summarize two points.

On the one hand, popularizing iGEM and synthetic biology via internet can reach a good effect because nearly half of these high school students said they heard iGEM and synthetic biology from the internet. We all think wiki is really a good platform to popularize synthetic biology and communicate with other iGEMers. What’s more, publishing more books relating to synthetic biology and iGEM is also a good conduit to let synthetic biology and iGEM known by other people, especially the young.

On the other hand, teacher and parents seem to have difficulties in accepting a new technology in China or in the whole world so that students can’t learn some interesting and impressive new science and technologies from adults. It’s very common in China but we, as a new generation, should try our best to realize the rapid changing of our society.

Future plans

Finally we drew some conclusion to improve our survey next time. In order to obtain results that are more valid we should launch a more widely circulated survey. In addition, perhaps more copies can be distributed at random to the general public. On the other hand, it’s really important to make an effective and smart questionnaire to get a more cogent result. Maybe we can make two kinds of questionnaire to hand out before and after the lecture to compare to get a better conclusion.

Besides those activities related to synthetic biology we’ve mentioned above, we assisted XMU_Software in finishing a theme fresco in Furong Tunnel.

Furong Tunnel can be said to be the most attractive scenery of Xiamen University, visited by more than 30,000 people per day statistically. We made this work in consideration of its attractiveness, which can greatly help us to popularize not only synthetic biology and iGEM but also our team. In fact, many students witnessed the fresco, maybe called doodles more appropriately, made by iGEMers from Xiamen University. Some of them took photos and asked us about iGEM. It improved a good interaction between our team and these students and tourists to popularize iGEM competition and synthetic biology.

First

We collaborated with Purdue iGEM team to create a definitive characterization standard for the parts registry. All the teams participated required to fill out a survey which about the important items in the registry page. Purdue iGEM team worked out a standard protocol which contains all of the details. The other teams need to complete it better or put forward some questions and problems. To make this work easy, we had a video meeting with the Purdue iGEM team and USP Brazil team though the GOOGLE HANGOUTS. It really helped us to exchange the opinions just like face to face.

As we know, the members of this collaboration are currently over 50 teams so that it is the biggest collaboration in the iGEM history. We feel awesome about what we did!

Second

On April 5th, 2013, Haoqian Zhang, advisors of Peking University iGEM team, a famous iGEMer who is keeping on develop synthetic biology and iGEM in China, come to XIAMEN University in purpose of strengthening collaborations our two universities. During his visiting, Mr. Zhang took part in discussions between our two teams, XMU_ Software and XMU-China2013 and gave some good advice focusing on the feasibility，security，advantages and disadvantages of ideas raised by our team members. His words about teamwork was very impressive and educational. Finally, Mr. Zhang simply showed some interesting projects in the past as well as encouraged us all to try our best to perform this year's iGEM projects, which greatly encouraged us.

Third

From June 24th to June29th, We provided parts BBa_C0061 and BBa_I0462 to the Toulouse iGEM team due to the high transform difficulty they described using this year’s distribution. Toulouse could directly use them for their cloning ,while we got some valuable feedback about our parts.

Fourth

In the summer vacation, we drew a doodle about 2013 iGEM in Furong tunnel in the camp with the member of software team of our university. This tunnel is not only a main stem but also a famous scenic spots, so that more than 30,000 people per day will pass by and look our doodle. In addition, the doodle is a quite novel approach to introduce synthetic biology and we believe that no team did it before.

We also held a garden party with software team where introduced iGEM and synthetic biology to 200 students of senior high school from the whole Fujian Province. Go to page…to see more

In the wet lab, we helped them to structure their plasmids, such as teaching how to ligation and digestion.

Fifth

We’ve also had a really good communication with the iGEM team of Peking University. From August 11th to August 19th ,one of our team members went to Peking University to get a deep cooperation with them. Firstly we introduced our own project in this year and exchanged the opinions .They gave us some good suggestions in experiments and models. What’s more, they gave us one BioBrick of sfGFP (BBa_...), which gave a great help to our structure. This sfGFP allowed our circuit to generated a brighter fluorescence and made easier to obverse in the microfluidics.

Sixth

We had a short meeting with 4 members of NJU iGEM team visiting us on June24th, 2013. We welcome all other visitors as well. During the meeting, we introduced our project this year to these visitors and exchanged some opinions with them. It can improve the connection and collaboration between different iGEM team in China.

We would like to extend our sincere gratitude to all those who helped XMU-China2013 during this year. Without your help we wouldn’t have completed this project successfully. Some pieces of our appreciation are as follows.

Team XMU-Software, the dry lab team of our university. Thank you for giving us so much help on wiki and modeling.

Graduates in Lab 571,Lab 580 and Lab 117, College of Chemistry and Chemical Engineering , especially Chiming Ye, Tingting Wu, Yu Zhang, Nan Wang and Jing Guo. Without your experimental guidance we wouldn't have managed to finish the whole project.

Prof. Hasty and his student Arthur Prindle, Department of Bioengineering, University of California, San Diego, La Jolla, California. Thank you for your patient and warm reply to our questions.

Prof. Ningshao Xia and his graduates, School of Life Sciences, Xiamen University, for providing us equipment and experimental guidance on electroporation.

Prof. Shoufa Han, Department of Chemistry, Xiamen University, for providing us fluorescence microplate reader and his students for experimental and equipment guidance.

Institute of Urban Environment, Chinese Academy of Sciences for providing us MALDI-TOF-MS.

Fujian Chemical Society and Prof. Yihui Chen. It's very kind of you to give us great support in Human Practice.

College of Chemistry and Chemical Engineering. You've got our six.

Xiamen University Academic Administration. Thank you for supporting us.

High school students from all over Fujian Province taking part in our Human Practice. Without your great support we couldn't make a successful Human Practice.

Peking iGEM 2013. Thanks for your sfGFP and some pieces of advice. If not we couldn't finish our work.

Finally, again we’d like to express my heartfelt appreciation to all these kindness and enthusiasm. Thank you. Thanks for your self-giving helps.