Team:Nanjing-China/Collaborations

Team Collaboration

1.Nanjing-China

This year we are cooperating with team SJTU-BioX-Shanghai. We come up with an idea of combining the two projects to achieve a win-win goal, which is to desalinate sea water with a high concentration of heavy metal. They are engineering with blue algea which can desalinate sea water while we work with engineered B. subtilis which can adsorb lead, gold and uranyl selectively. We also sent them our cell adhesion materials in case they need to separate blue algea from sea water. They have been trying to utilize the material to optimize their project.

Fig 1. Cell adhesion materials. 1. First generation plastic pellet 2. Second generation plastic pellet 3. Cyclic fiberfill 4. Chain-like fiberfill

Firstly, as is known, different organisms live under diverse conditions. Therefore the first problem that we meet with is related to the living conditions of our chassis. Can our engineered B. subtilis survive in sea water? Can their blue algea survive in aquatic environment with high solution of heavy metal? Only if we solve the two obstacles can we cooperate with each other smoothly. So we decided to do some tests to confirm whether they can survive simultaneously in sea water with heavy metal. We planned to select microorganisms that meet the collaboration requirement. Unfortunately, the microorganisms used in our project were both chosen. It could be unpredictable if we had tried to modify them. Thus we had to change our plan.

After many rounds of discussion, we found that places such as estuaries could provide the two organisms with required environment. So we finally decided to desalinate polluted sea water step by step. Sea water with high concentration of heavy metal go through our purification device first so that heavy metal contaminants can be adsorbed. Next, blue algea functions to desalinate the sea water. The advantage of our idea is that we only need to make sure that our engineered B. subtilis can survive in sea water while the low efficiency problem is also inevitable.

We have been collaborating with each other since early July and the collaboration is still going on. We sincerely hope that our win-win goal can be achieved some day. Because we have found that almost all desalination technologies encounter the problem of sediments which contained a variety of heavy metal such as lead. The combination of our two teams not only identified the significance of collaboration, but could solve some real-life problems as well.

2.SJTU-BioX-Shanghai

This year, we were very close with Nanjing-China team. They are dealing with heavy metal problems through biofilms. When we talked about collaboration, we first thought about binding two projects on seawater desalination field. Thus, we discussed an application with this two project combining. Our team is responsible for explain this application and its supporting fact. Nanjing-China team will explain the details which needs to be discussed to make this working for real.

First, we start with the features of seawater and the end purpose of the combination project. Most of the seawater desalination plant use open-water intake near costal to avoid intake costs. This also means that the factories can’t avoid to deal with different pollutions in costal seawater, including heavy metal pollution.

The main source of coastal heavy metal pollution is runoff and atmospheric sedimentation. From 2006 to 2007, Oceanic Administration of People’s Republic of China did a massive-scale offshore detection for water condition. In its report, the concentration of lead, copper, zinc and mercury in seawater which is over the first seawater class at some monitoring spots. Among them, average lead concentration in the Bohai Sea is over the first seawater class, and some of the monitoring spot detect a number over the second seawater class. The natural degradation of heavy metal is very difficult. Also, the offshore is relatively big water body which makes the pollution hard to increase as well as decrease. Overall, the heavy metal pollution of Chinese offshore cannot be overlook when we use seawater as material.

In normal desalination process, even normal resource water has to go through some sedimentation processes. In fact, one of the safety concern of general seawater desalination is what do we do with those sediments. The idea of cooperating two project is to merge Nanjing-China team’s project to achieve a new seawater recovery and utilizing system.

The word “circular economy” should be familiar to most people. In China, this is one of the guiding ideology for the economic and society development. The core of the concept is to maintain the balance between resource and waste. To balance the rapid consumption of resource, man should not only depend on nature but also ourselves to turn wastes into resources.

Unlike water has a strong nature balance itself, heavy-metal’s nature circulation is much slower. We need to use man power to turn every waste kind into resource. In this concept, seawater desalination combing heavy-metal recycling will be the key to build the heavy metal’s balance.

The origin of heavy metal comes from the river, therefore, it is hard to control from the origin. Most of the study dealing with heavy metal in seawater focus on recovery and has to work in an open condition. The efficiency of absorption largely depends on the mobility of seawater. IT also brings some safety concern by introducing new species to original ecosystem.

For example, the main recovery method focuses on mangrove in China. Mangrove is salt-torrent and absorb heavy-metal naturally. However, because it is plant, the study and application of mangrove will be relatively slow.

Seawater desalination is one of the few seawater alternating technology that pumps in the seawater. It also has complicated process, and part of it could be replaced by Nanjing’s project. Thus, we should look at two projects’ combination as environmental restoration and seawater alternation using the same artificial water flow.Back to circular economy, this will increase the force that heavy-metal waste change into resource with a low cost adding.

Journal

In March, we founded our team and recruited our team members, after with we divided our team members into several groups and each group is responsible for one part of the competition. We were also busy brainstorming about our project and signing up for iGEM competition.
In April, we confirmed our project. A majority of the team members wanted to deal with problems related to heavy metals. Then we searched online and read many articles related to our project. We also made a plan about the procedures of all the experiments.
In May, we were busy with kit construction and part construction. We were also contacting different authorities and scheduled for interviews.
In June, we went on with kit construction and started the homologous recombination part. We also interviewed with a government official.
In July, we were engaged in adhering the engineered bacteria onto the material that we used. We also tested for the four kinds of bacteria adhesion materials. Some members were busy with modeling. We also started our collaboration with Shanghai Jiao Tong University.
In August, we were doing experiments about heavy metal adsorption and busy with the test of our designed device. We also attended the CCiC conference in Beijing and presented our project to Chinese iGEM teams. Besides, we also interviewed with a college professor, an advisor in a sewage disposal enterprise and an organizer in an environmental protection group.
In September, we were preparing for the Jiant Jamboree in Boston, including the posters, wiki, leaflets and mascots.

Protocols

1.DNA purification/Axygen gel extraction

Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination.
Absorb the liquids left on the surface of the gel slices using paper towels. Weigh gel slice (tare with empty tube).
Add 3 volumes of DE-A buffer per mg of gel (so a 100mg gel gets 300ul of buffer).
Resuspend the gel in Buffer DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved (keep heating for 6-8 minutes). If low-melt agarose gel is used, you may heat it at 40℃. Intermittently vortexing every 2-3 minutes will do a lot of help to accelerate the solubilization.
Note: Buffer DE-A is red liquid, so you can observe the color to make sure the gel is fully dissolved.
Add 0.5× Buffer DE-A volume of Buffer DE-B and mix. If the DNA fragment is less than 400bp, supplement further with a 1×sample volume of isopropanol.
Note: After the addition of DE-B, the solution should be in the uniform color of yellow.
Place a Miniprep column into a 2ml microfuge tube (provided). Transfer the solubilized agarose from the step above into the column. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Return the Miniprep column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.
Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.
Place the Miniprep column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Place the Miniprep column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.
Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 50ul of ddH2O to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.
Note: Pre-warm the ddH2O at 65℃ will generally improve elution efficiency.

2.Preparation of chemically competent E.coli cells

Inoculate 2ml LB broth with an aliquot (about 50ul)of the desired E.coli from the -80℃ freezer stock of cells.
Incubate for 2h at 37℃.
Add the 2ml seed culture to 250ml LB broth and grow at 37℃, shaking (about 200rpm) until OD600 of 0.3-0.4 (about 5 hours).
Pre-cool the 50ml polypropylene tube, 80 EP tubes, CaCl2-glycerine (0.1mol/L CaCl2) and CaCl2- MgCl2 (80mmol/L MgCl2, 20mmol/L CaCl2). Set the centrifuge and prepare the ice tray.
Transfer the bacteria into the 50ml polypropylene tube. Place it on ice for 10 minutes.
Centrifuge at 4℃, 4100rpm for 10 minutes.
Discard supernatant, then place the tube upside down to make sure trace liquid medium runs out.
Add 30ml of pre-cooled CaCl2- MgCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.
Centrifuge at 4℃, 4100rpm for 10 minutes.
Discard supernatant then place the tube upside down to make sure trace liquid medium runs out.
Add 2ml of pre-cooled CaCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.
Transfer to EP tubes (50ul every tube) and store at -80℃.

3.General Heat-Shock Transformation

Add 10ul DNA to 50ul cells on ice (set positive control by using Pcotc, cotc, PtasA, GolB,PbrR DNA fragment and ddH2O, set negative control by using chemically competent E.coli cells without plasmids).
Incubate on ice for 30 minutes.
Heat shock at 42℃ for exactly 90 seconds.
Place samples back on ice for 1-2 minutes.
Operating in the clean bench, add 900ul of LB broth per tube.
Incubate at 37℃ for 60 minutes, shaking.
Activate it on the plate for 60 minutes. The total number of plates is 7.
Centrifuge at 3000rpm for 1 minute.
Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells.
Use the inoculating loop to load bacteria liquid then streak on the LB plate.
Place plates upside down and incubate at 37℃ overnight.

4.PCR method/Taq PCR

Thaw Taq, dNTP, primers, template DNA (pcotc, cotc, PtasA, tasA, GolB, PbrR) on ice.

To a new PCR tube, add:

template DNA	1ul
dNTP	1ul
10×buffer	5ul
Mg2+	3ul
F primer	1ul
P primer	1ul
rTaq E	1ul
ddH2O	37ul
total	50ul

Mix solution well.
Place tube in PCR thermocycler. Set thermocycler program:
Inititial denaturation: 3min at 95℃;

Loop (29 cycles), Denaturation: 30s at 95℃,Annealing: 30s at 60℃,Elongation: 1min at 72℃;

Final elongation: 10min at 72℃;

Store: 12℃.(not for too long).
We use 5ul of the PCR product for electrophoresis and 45ul for purification (details see DNA purification/AxyPrep PCR DNA purification PCR).

5.DNA purification/AxyPrep PCR DNA purification PCR

Add 3 volumes of Buffer PCR-A to the solution (if Buffer PCR-A is less than 100ul, then add to 100 ul). Mix gently and then transfer to a Miniprep column, which is placed in a 2ml microfuge tube (provided).
Centrifuge at 12,000rpm for 1 minute and discard the filtrate from the 2ml microfuge tube.
Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Return the Miniprep column to the 2ml microfuge tube and add 400ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Note: this step can be omitted.
Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 25-30ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.
Note: Pre-warm the Eluent or deionized water at 65℃ will generally improve elution efficiency.

6.Plasmid extraction

Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely.
Add 250ul of Buffer S1 to the pellet to resuspend bacteria cells.
Add 250ul of Buffer S2, mix gently by inverting the tube 4-6 times until the solution becomes clear. The time should be no longer than 5 minutes.
Add 350ul of Buffer S3, mix gently by inverting the tube 6-8 times.
Centrifuge at 12,000rpm for 10 minutes.
Place spin column into a 2ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Return the column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Return the column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Place the column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.
Transfer the column into a clean 1.5ml microfuge tube (provided). Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.
Note: Pre-warm the Eluent or deionized water at 65℃ will generally improve elution efficiency.

7.Agarose Gel Electrophoresis

Weigh agarose powder and TAE buffer according to a proper portion, and add them to a 100ml conical flask (we usually make 1.5% Agarose Gel).
Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave).
Let the solution cool down to about 40-50℃ and add DNA gel stain (usually we use EB), pour the solution into the gel casting tray with appropriate comb.
Let the gel cool until it becomes solid.
Pull out the comb carefully.
Place the gel in the electrophoresis chamber.
Add enough TAE Buffer so that there is about 2-3mm of buffer over the gel.
Pipette DNA samples mixed with appropriate amount of DNA loading buffer (the dye/GeneFinder is in the loading buffer) into wells on the gel.
Run the gel at 135V for about twenty minutes.

8.Colony PCR

We carry out colony PCR in order to amplify a few copies of DNA across several orders of magnitude and check the length of DNA sequences between two designed primers.

To 20 new PCR tubes (add 2ul bacteria cells and operate thermal cracking at 95℃ for 15 minutes), add:

template DNA	2ul
dNTP	1ul
10×buffer	5ul
Mg2+	3ul
F primer	1ul
P primer	1ul
rTaq E	1ul
ddH2O	36ul
total	50ul

To 2 new PCR tubes (tasA, pbrR), add:

template DNA	1ul
dNTP	1ul
10×buffer	5ul
Mg2+	3ul
F primer	1ul
P primer	1ul
rTaq E	1ul
ddH2O	37ul
total	50ul

Mix solution well.
Place tube in PCR thermocycler. Set thermocycler program:
Inititial denaturation: 3min at 95℃;

Loop (29 cycles), Denaturation: 30s at 95℃,Annealing: 30s at 60℃,Elongation: 1min at 72℃;

Final elongation: 10min at 72℃;

Store: 12℃.(not for too long).
use the PCR product for electrophoresis.

9.Protocol for restriction enzyme digestion

To a 1.5ml microfuge tube (Ptt, Pcc), add:

Insert	17ul
bamHⅠ	1ul
KpnⅠ	1ul
10×K	1ul
total	20ul

10.Protocol for DNA ligation

To a 1.5ml microfuge tube (Ptt), add:

Vector	1ul
Insert	7ul
T4 Buffer	1ul
T4 Ligase	1ul
total	10ul

To a 1.5ml microfuge tube (Ptt control), add:

Vector	1ul
ddH2O	7ul
T4 Buffer	1ul
T4 Ligase	1ul
total	10ul

To a 1.5ml microfuge tube (Pcc), add:

Vector	1ul
Insert	7ul
T4 Buffer	1ul
T4 Ligase	1ul
total	10ul

To a 1.5ml microfuge tube (Pcc control), add:

Vector	1ul
ddH2O	7ul
T4 Buffer	1ul
T4 Ligase	1ul
total	10ul

Incubate at 16℃.

11.Preparation for competent B. subtilis cells

Streak out the strain to be made competent on an LB or TBAB agar plate as a large patch and incubate overnight at 30 ℃.
The following morning scrape off the cell growth off the plate and use to inoculate fresh, pre-warmed, SpC medium to give an OD600 reading of about 0.5.
Incubate the culture at about 37℃ with vigorous aeration and take periodic OD readings to assess cell growth.
When the rate of cell growth is seen to depart from exponential inoculate 200ml of pre-warmed, SpⅡmedium with 2ml of stationary-phase culture and continue incubation at 37℃ with slower aeration
After 90min incubation, pellet the cells by centrifugation at room temperature.
Carefully decant the supernatant into a sterile container and save.
Gently resuspend the cell pellet in 18ml of saved supernatant and add 2ml of sterile glycerol; mix gently.
Aliquot the competent cells in sterile tubes, freeze rapidly in liquid nitrogen or a dry-ice bath and store at -70℃.

12.Transformation

Thaw competent cells rapidly by immersing frozen tubes in a 37 ℃ water bath.
Immediately, add one volume of SpⅡEGTA to the thawed cells; mix gently.
In a sterile test tube add competent cells to the DNA solution and incubate in a roller drum at 37℃.
Dilute the transformed cells as appropriate in T Base containing 0.5% glucose and plate immediately onto selective media.

13.Amylase activity analysis

Apply iodine on the plate with 0.1% starch and spectinomycin.
Incubate the engineered bacteria and non-engineered bacteria onto the plate at 37℃.
See if there are hydrolysis circles on the plate.