Team:WHU-China/Notes

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Materials for cloning all the genes

Bacteria strain:

E.coli DH5α

Minimal Medium:

M9:

For 1L Medium add

Na₂HPO₄·12H₂O 15g

KH_{2PO4 3g}

NaCl 1g

NH_{4Cl 0.5g}

After autoclaving, add:

MgSO_{4(1mol/L) 2mL}

Bacteria strain:CaCl_{2 100μL}

Triton X-100 (as emulsifier) 2uL

Note：

1.For M9 medium using oleic acid as sole carbon source, various amount of oleic acid was first emulsified 1:1 with 10% Triton X-100. M9 medium was then slowly added with constant vortex. M9 medium with high concentration of oleic acid was diluted by M9 medium with triton to form various concentrations；

2. For M9 medium using glucose as sole carbon source, M9 medium with high concentration of glucose was diluted by M9 medium to form various concentrations.

Step:

1. Seed liquor which was activated over night was inoculated into M9 medium which contains different concentration of oleic acid. And it was then incubated at 37℃ for 24 hours;

2. After 24h of incubation in 24 well plates in 37℃, bacteria culture was centrifuged at 3000rmp for 5min, washed and resuspended in PBS;

3. We detected the OD600 and fluorescence of using SpectraMax M2 plate reader (Molecular Devices) .Excitation at 584 nm and emission at 607 nm were used. All fluorescence was normalized with cell density by measuring the absorbance at 600 nm.

Result:

Normalized using Fluorescence/0D600

Blue: Constitutive promoter J23110

Red: PfadR

Glucose Concentration gradient: 0.5, 1, 5, 10 mM

Fatty acid Concentration gradient: 0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5 mM

this part is finished

Protocols of Cupric-Soap Reaction:

To test metabolism of long fatty acids, we used oleic acid as sole carbon source in mediums, and used cupric-soap reaction to determinate oleic acid concentration preliminarily.

Modified M9 minimal medium with emulsified oleic acid as sole carbon

Minimal medium was the same with that in Materials and methods for PfadR

What's Important:

Slowly pour M9 minimal medium into mixture of oleic acid and Triton X-100 to get more homogeneous solution.

Analysis the concentration of the oleic acid in the medium by cupric-acetate method

1.Collect 5 ml medium which has been used to cultivate bacteria. Then centrifuge the medium at 3000rpm for 10 min to separate bacteria and medium;

2.Decant 3ml supernatant liquid into a 10ml EP. Add 3ml acetone to the liquid, 1ml at a time, shaking 10-20 times before adding another 1 ml in order to avoid the effect of the ions of the liquid during the extraction progress;

3.Add 3 ml isooctane once ,shaking for at least 90s, stand for 2min or longer until layering completely;

4.Collect 3 ml clear isooctane in a 5 ml EP, Add 800l cupric-acetate (5% m/v, adjust pH to 6.8 with pyridine), Shaking for 90 seconds, stand for 2min or longer until layering completely;

5.Detect OD of organic phase in a spectrometry at 715nm.

The Standard Curve:

We add 1.6ml, 3.2ml, 4.8ml, 6.4ml, 9.6ml, 11.2ml oleic acid into 3ml M9 medium to generate a gradient. The absorbency is measured as described in the protocol.

We slightly modified the methods provided by Kwon and Rhee, 1986, adding the extraction step, for it is dispensable when bacteria are in the medium. However, the standard curve is still very close to the paper’s, which proves that plausible and accurate

Krebs-Ringer phosphate（KRPD buffer solution）：

Solution A：

For 100 mL:

NaCl 7.5985g ; KCl 0.3727g ; CaCl2 0.1054g; glucose-H2O 0.9495g,

For 100 mL:

NaH2PO4·2H2O 0 .2964g ;Na2HPO4·12H2O 2.9011g，

After dissolving, add MgSO4·7H2O 0.3130g

Mix A solution and B solution, add 790ml dd H2O, then regulate pH to 7.2~7.4 using 1M NaOH/1M HCl, finally add dd H2O to a final total volume of 1L.

Procedures

1. cultivate cells in 1 liter of medium to late exponential phase;

2. harvest by centrifugation at 35℃;

3. wash twice with 2 liters of warm(35℃)0.05M potassium phosphate buffer (pH 7.4) containing 1%(v/v) Triton X-100;

4. resuspend in 0.05M potassium phosphate buffer (pH 7.4) containing mercaptoethanol;

5. add sufficient volume of buffer to give a concentration of about 50mg(dry weight) of cells/ml;

6. disrupt cells with a Bransor Sonifier for 1min. The treatment was applied for 15s intervals, under 4 ℃;

7. centrifuge at10,000×g for 30min;

8. decant supernatant liquid for enzymatic assay, the protein concentration was determined by the biuret method;

9.analyze fatty acid oxidation in cell-free extracts

Note: little oxidation was observed when less than 1mg.

Reaction mixture:

1.0ml of freshly oxygenated Krebs-Ringer phosphate(pH 7.4)

Palmitate-1-14C, 20nmole (80,000 counts/min)

CoA, 1 μ mole

NAD, 1μ mole

ATP, μ mole

Succinate, 10 n moles

Supernatant protein, 1~5 mg

dd H2O to a final total volume of 2.0ml

Cellulose biosynthesis

1. Inoculate the bacteria into liquid LB medium and then incubate it for 24 hours at 37℃;

2. After centrifugation, supernatant is preserved for use, while deposits are resuspended with PBS and adjust to OD600 1.0;

3. Exceed cellulase was used to digest cellulose in supernatant and deposits. After the cellulase is added, 1mL solution was sampled every 3 min and cellulose of the sample was inactivated immediately;

4. Ten minutes later, the reduce sugar is detected in all the samples with or without cellulose;

5. 2mL DNS solution is mixed with the sample, and incubated in boiling water for two minutes, then it is cooled rapidly;

6. After 9 mL ddH2O being added into the solution, record absorbance at 540nm;

The Standard Curve of glucose concentration

Materials and methods of SDS-PAGE:

Coomassie Blue Stain

-Coomassie brilliant blue G-250 50mg

-95% ethanol 25ml

-85%H3PO4 50ml

-H2O, adjust to 500ml

-filter

Destain solution

-methanol 250ml

-acetic acid 50ml

-H2O adjust to 500ml

2x SDS loading buffer

-0.5mol/l Tirs-HCl(PH6.8) 25ml

-10%SDS 8ml

-50%glycerol 20ml

-2-mercaptoethanol 2ml

-1%Bromphenol Blue 4ml

-H2O adjust to 100ml

10xSDS-PAGE running buffer

Tris base, 30.3 g

M glycine 144.1g

SDS 10 g

-H2O adjust to 1L

Steps

Protein expression

1.inoculate the liquid strains into LB medium supplement with 50μg/mL ampicillin, incubate the medium at at 37℃ until OD600 reaches 0.6;

2. Separate the culture into two test tubes. Add IPTG into one of two tubes at a final concentration of 1mM to induce the expression of

3. 1 mL of the culture is sampled every hour. After centrifugation, deposits was suspended with 300 μL PBS and 200 μL 2x SDS loading buffer, then incubated in boiling water for 15 min.

4. 10μl of samples was load in lanes, run the SDS-PAGE

5. Stained the SDS-PAGE 5 hours

6. Destain the SDS-PAGE until you can see the protein band.

team history

Dec. 2011

WHU iGEM team was established

Dec. 2011 to Feb. 2012

Every one presented their own idea, then we discussed the feasibility of these ideas.

Feb. 2012

The final project was determined, named E.coslim

Mar. 2012

We finished an outstanding presentation. Our reply was approved by the leaders of college of life sciences, Wuhan University.

Apr. 2012

Our iGEM team was divided into 3 groups—group of Sheng, group of Xia and group of Mei.

Apr. 2012 to prsent

Experiments started….

Apr. 2012

Group of Sheng: FADR was connected to pSB1A2 plasmid carrier

FADR was connected to RFP gene

Group of Xia: starting to connect genes of CI control system

Testing the function of CI control system

Group of Mei: YhjH/ FadE/ FadD/ FadL gene was connected to pSB1A2 plasmid carrier

May. 2012

Group of Sheng: FadB/ FadJ gene was connected to pSB1A2 plasmid carrier, then BBa_B0030(RBS) as well

Group of Xia: They devised two promoters p110 and p101, which were expected to be controlled by glucose. However, they failed.

Group of Mei: YhjH/ FadE/ FadD/ FadL gene was connected to BBa_B0030(RBS)

June 2012

G of S: FadR was connected to low-copied plasmid carrier.

sFadB and sFadA genes were connected to BBa_B0030 (RBS)

sFadE gene was connected to pSB1A2 plasmid carrier

G of X: the connection of genes, which were relative with cellulose, was finished.

Another two promoter were devised, p1 and p2

G of M: YhjH/ FadE/ FadD/ FadL gene was connected to BBa_B0024 (terminator)

July 2012

G of S: FadJ was connected to BBa_B0024 (terminator), sFadE gene was copied by PCR technology

On the front half of this month, they conducted several pre-experiments of oleic acid test

On the next half month, they started normal experiments of oleic acid test

G of X: started to test the function of p1 and p2

G of M: YhjH/ FadE gene was connected to BBa_J23100 (promoter)

Finish the standard curve of oleic acid test

Aug. 2012

G of S: sFadE was induced to point mutation, then it was connected to BBa_B0030 (RBS) and BBa_B0024 (terminator) respectively

sFadB was connected to BBa_B0030 (RBS) and BBa_B0024 (terminator) respectively.

G of X: test the function of cellulose-controlled genes, having got satisfying results

G of M: copied Adra gene and finished the connection of BBa_B0030 (RBS), BBa_B0024 (terminator) and BBa_J23107 (promoter), BBa_J23114 (promoter) respectively. FadE/YhjH/FadD/fadL were connected to BBa_J23107 (promoter), BBa_J23114 (promoter).

Sep 2012

G of S: Transferred all the parts in pSB1A2 into pSB1C3, Tested the function of PfadR, Characterized the effect of each gene on fatty acid consumption, started to set up the platform for in vitro experiments.

G of X: Transferred all the parts in pSB1A2 into pSB1C3, tested the function of cellulose system. Repeat the test of the function of cellulose-controlled genes.

G of M: Transferred all the parts in pSB1A2 into pSB1C3, test the function of Adra/YhjH.

About E.coslim by Kuanwei Sheng

In order to help people lose weight, besides our three devices, we also have come up with many other creative ways.

Ⅰ.Short chain peptides synthesis

Recent researches have indicated that some short chain peptides in intestine have effect on inhibiting appetites, therefore decrease the food intake. We thus formed the idea that we could synthesis a DNA chain that encodes those short peptides.

Ⅱ.Biosynthesis of L-carnitine

L-carnitine is a molecule that facilitates the progress of transporting fatty acids into mitochondria where these fatty acids will be disintegrated. We once considered use L-carnitine to help the host metabolize fatty acid better. However, the biosynthesis of L-carnitine has too many derivatives or the pathway is patented by others.

Ⅲ.Xylose isomerase

Xylose is a prebiotics that can hardly be absorbed by human. We consulted many papers and find that glucose can be converted into xylose by xylose isomerase. Therefore, we thought maybe we could lower the glucose available in intestine by expressing xylose isomerase. However, this process is shown to be reversible latter. Mutated the sequence of the protein may generate high converting rate, yet it is too laborious and risky for a short time project.

Other novel ideas

Tackle Water bloom by Tong Wang and Kuanwei Sheng

Since the detrimental effects caused by cyanobacteria to the environment such as making water carcinogenic have become a serve global problem, we therefore tried to employ E.coli as an expression system to eliminate these detrimental effects. When we first took over this project, we thought about limiting the growth of cyanobacteria. Along with the process we got to read a large amount of relevant papers about cyanobacteria, we found that not only the main detrimental effects caused by cyanobacteria is attributed to its product which is a cyclic peptide called microcystin, but also microcystin can regulate the population density. Then we came to realize the importance of microcystin and began to search information about it. Through over this process, we discovered a gene cluster which is responsible for the microcystin degradation pathway. It encodes four enzymes——MlrA、MlrB、MlrC and MlrD. Also, we found that some non-toxic cyclic peptides produced by cyanobacteria such as Anabaenopeptin B and Anabaenopeptin F can induce lysis of cyanobacteria. The latter finding can be utilized as an effective cell population density control mechanism. Thus we thought about constructing two independent systems to eliminate cyanobacteria, one about inducing lysis of cyanobacteria and the other about degrading microcystin. Finally we gave up this project because of the reasons that these gene clusters are too large to be expressed in E.coli and that E.coli cannot survive in the sea, however, we still feel proud of these fancy ideas.

Desalination of sea water by Kuanwei Sheng

We came up with the thought that engineering bacteria can intake ions like Na+, cl-, Mg2+ and etc. under special stimulus. Also, under another certain stimulus, the bacteria can export those salts out of cells for reuse. Therefore, we can use these bacteria to desalinize sea water and extract the salt the same time. However, there is no such ion channel that can meet our needs.

Sense the earthquake By Min Ye

We once tried to find proteins that can sense vibration and construct a pathway to report that vibration. However, we are not able to find the protein that can meet our needs.

Auto plasmid preps By Kuanwei Sheng

We thought about constructing a synthetic protein that combines zinc finger protein which can recognize the specific sequence of DNA and signal peptide that can make proteins be exported out of the bacteria by secretion pathway. Therefore, it is possible that plasmid can be exported out the cells together with the zinc finger protein.

Outer Membrane Vesicle (OMV) to treat cancer By Kuanwei Sheng

We thought to use Outer Membrane Vesicle (OMV) to treat cancer. Specifically, we thought about localizing antibody that can recognize certain cancer on the surface of OMVs via signal peptides. Also, we thought we may localize cell division inhibitor protein or protein that lead to cell death inside the OMVs. Therefore, we hoped that the OMV excreted by the bacteria can recognize and kill cancer cells.

Multicell Yeast By Wenxiong Zhou

Transform the yeast from a single-cell microbe to a multi-cell organism by setting bistability of gene expression among cells of yeasts adhered in amalgamation.