Team:ZJU-China/notebook.htm

This section is a summary of some outstanding ideas we developed during our brainstorming stage in the beginning two months after our team was founded.

List

Controlled chemistry plant

Synchronized bacterial clock

EPad

Methane alarm

Silkworm

Radio-communication

Plant doctor

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Controlled chemistry plant

As we know, biofilms are always used for chemical transformations in biorefineries. To utilize biofilms more efficiently, we need to control them and make them replaced. Here we expect to use dispersal proteins along with population-driven quorum-sensing switches to accomplish this goal.

We found some paper on related studies. In one of them, the researchers form an initial colonizer biofilm in a microfluidic device, introduce a second cell type called disperser into this existing biofilm, form arobust dual-specied biofilm and displace the initial colonizer biofilm with an extraceller signal from the disperser cells. They also remove the disperser biofilm with a chemically induced switch, and the consortial population could tune. The sketch followed show the mechanism of it.

The two E.coli cell types communicate by using the LasI/LasR QS module of P.aeruginosa. In the disperser cell, the LasI protein(autoinducer synthase) is constitutively produces and synthesizes the QS signal 3oC12HSL. 3oC12HSL freely diffuses into the initial colonizer cell and makes a complex with LasR(LuxR family transcriptional regulator), and the 3oC12HSL+LasR complex induces biofilm dispersal protein BdcAE50Q by activating the lasI promoter. BdcAE50Q disperses biofilms by binding cyclic diguanylate. The biofilm dispersal protein Hha13D6 in the disperser cell is induced upon adding IPTG. Hha13D6 disperses biofilms by activating proteases.

(From "Synthetic quorum-sensing circuit to control consortial biofilm formation and dispersal in a microfluidic device")

We want to develop the system and found a system contains three E.coli cell types, in which the first one can be replaced by the second one, the second one can be replaced by the third one and the third one can be replaced when added some chemical signals. Thus, this kind of controlled chemistry plant can be applied to a three-step chemical reaction and each kind of cells will finish one of them in sequence, just as follows.

Reference:

1. "Synthetic quorum-sensing circuit to control consortial biofilm formation and dispersal in a microfluidic device"

(http://www.nature.com/ncomms/journal/v3/n1/full/ncomms1616.html)

2. "Engineering microbial consortia: a new frontier in synthetic biology"

(http://www.ncbi.nlm.nih.gov/pubmed/18675483)

3. "Applications of quorum sensing in biotechnology"

(http://www.springerlink.com/content/j16470w7g3838851/)

4. "A synthetic Escherichia coli predator-prey ecosystem"

(http://www.nature.com/msb/journal/v4/n1/full/msb200824.html)

Synchronized bacterial clock

Human orchestrate their activities in different places because of their synchronizing clocks. Isn't it cool if bacteria can coordinate their molecular timepieces in the same way as we human do?

We found that Hasty and colleagues have constructed a network of genes and proteins in E.coli that acts as a molecular clock and can be synchronized across a population of the bacteria. The mechanism can be described briefly as follows.

A promoter(PluxI)drives the production of LuxI, an enzyme that synthesizes the quorumsensing signal acyl-homoserine lactone (AHL). Another PluxI control ths the production of AiiA, a protein that catalyses the degradation of AHL, and a third PluxI triggers the synthesis of a variant of green fluorescent protein called yemGFP. An AHL receptor, LuxR, is constitutively expressed. The authors combined these components to form an autoinducng circuit (AHL activates LuxR, and the AHL-LuxR complex induces PluxI-driven luxI transcription and yemGFP production) with a time-delayed negative feedback loop (the AHL-LuxR complex induces PluxI-driven production of AiiA, which degrades AHL). The result was a population of bacteria that produce synchronized pulses of fluorescence, coordinated by quorum sensing.

(From "Synthetic biology: synchronized bacterial clock")

We expect to apply the network to medical field. Since the network can make an oscillation, it can be used to release medicine periodically. What is more, we wish to add another sensor in the network. The sensor can sense a signal which is released because of a certain disease, and thus the cells can sense the signal and start the oscillation which releases the medicine periodically. For instance, when a man hasdiabetes, the high density blood sugar will start an insulin-releasing oscillation.

Reference:

1. "Synthetic biology: synchronized bacterial clock"

(http://www.nature.com/nature/journal/v463/n7279/full/463301a.html)

2. "A synchronized quorum of genetic clock"

(http://www.nature.com/nature/journal/v463/n7279/abs/nature08753.html)

3. "bacteria collaborate to sense arsenic"

(http://www.nature.com/nature/journal/v481/n7379/full/481033a.html)

EPad

Ipad is popular all over the world, but how about an Epad? Have you thought that there is a tablet whose screen is made up of E.coli and it can sense your touch to display different colors?

MscL is a kind of membrane protein of E.coli. They control the transportation of matter, but they are usually closed. When the balance between intracellular and extracellular is broken, or the cytomembrane senses some unusual mechanical force, they will open and let the pressure disappear. We hope to utilize the protein and found a network which can display fluorescence of different color according to the state of MscL. In this way, a pressure-sensing and -controlling screen made of E.coli can be built.

Reference:

1. "Release of content through mechano-sensitive gates in pressurized liposomes"

(http://www.pnas.org/content/107/46/19856.short)

2. "Mechanical force and cytoplasmic Ca2+ activate yeast TRPY1 in parallel"

(http://www.springerlink.com/content/xg2q0601j73q1r31/)

Methane alarm

The concentration of methane in the coal mine is an important indicator which affects the life safety of miners. There should be alarm when the concentration of methane is between 1.0-1.5percent in the coal mine under the Chinese law. We aim to design a methane alarm for coal mine by methanotrophs, which consists of four parts: decomposition, sensor, signal tuner and output. Decomposition part takes in methane and conducts catalytic decomposition of methane to CO2 or assimilates methane as its own organic compounds. This function is the naturally exist in methanotrophs. Sensor senses the concentration of methane and produces activator A. As for signal tuner part, we plan to find a substance B which is sensitive to the concentration of activator A. Activator A and substance B combines to control the activity of substance C. Substance C acts as the switch of promoter--when the concentration of methane has been exceeded the alarm threshold, promoter will turn on. Output part will give the alarm by producing pigment of red lycopene when promoter is open. We plan to put methanotrophs on the miner lamp. When the bacteria produce pigment, the light through bacteria will turn out to be red.

Reference:

1. "Biological Methane Oxidation: Regulation, Biochemistry, and Active Site Structure of Particulate Methane Monooxygenase"

(http://informahealthcare.com/doi/full/10.1080/10409230490475507)

2. "Importance of methane-oxidizing bacteria in the methane budget as revealed by the use of a specific inhibitor"

(http://www.nature.com/nature/journal/v356/n6368/abs/356421a0.html)

Silkworm

Hangzhou is famous for silk. We wish to let engineered bacterial produce silk proteins, just like lovely silkworm. A machine which can make threadlike things with the proteins will be used to make silk.

Reference:

1. "Morphology and primary crystal structure of a silk-like protein polymer synthesized by genetically engineered Escherichia coli bacteria"

(http://onlinelibrary.wiley.com/doi/10.1002/bip.360340808/abstract)

2. "Biotechnological Production of Spider-Silk Proteins Enables New Applications"

(http://onlinelibrary.wiley.com/doi/10.1002/mabi.200600255/full)

Radio-communication

Synthetic biology always needs bacteria to sense an input signal and output another. It occurs to us that we can use two kinds of bacteria to accomplish the task. The first one kind of bacteria senses the input signal and then fluoresces, while the second one senses the fluorescence from a distance far away from the first one and then output the final signal. It do not need a straight contact between the two kinds of bacteria, seeming like a radio-communication.

Plant doctor

When the leaves of some kinds of plants are bitted by insects, the substance A in the juice of plants will react with the one B in the spit of the insects. As a consequence, a volatile matter is produced, which attract the natural enemy of the insect. We plan to engineer the bacteria, having a symbiosis with plants, with genes of the enzymes which catalyze the production of the substance A in the juice. The bacteria will make plant doctors.

Week 1 (06.22-06.24): Preparation

June 22

1. Do some washing.

2. Prepare LB plates, including ampicillin, kanamycin and spectinomycin resistance.

3. Get some E.coli DH5αcompetent cells from Prof. Xiao Mu. Receive E.coli BL21*(DE3) strain requested previously.

4. Transformation: extract plasmid pCJDD0 (Ampr), pCJDFA (Kanr) and pCJDFB (Spr) from filter paper received, transform them into DH5αcompetent cells respectively.

June 23

Amplify E.coli BL21*(DE3) strain.

June 24

1. Make glycerol stock of E.coli BL21*(DE3) strain.

2. Make competent cells of E.coli BL21*(DE3) strain and DH10β.

Week 2 (06.25-07.01): Amplify pCJDD0, pCJDFA and pCJDFB

June 25

Pick up single colony of pCJDD0 and pCJDFA from plate of June 22 and amplify them in liquid LB medium.

June 26

1. Transform pCJDD0 (Ampr), pCJDFA (Spr) and pCJDFB (Kanr) into DH5α and DH10β respectively and adjust the resistance from that of June 22 because of a small mistake in supplementary information of reference.

2. Miniprep plasmid of pCJDD0 from bacteria of June25.

Name: D0-6-26-1 Concentration: 27.5 ng/ul A260/280: 1.76

Name: D0-6-26-2 Concentration: 24.7 ng/ul A260/280: 1.81

3. Transform plasmid D0-6-26-2 into DH10β

June 27

1. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH10β), pCJDFB (DH10β) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.

2. Miniprep plasmid of D0-6-26-2 from bacteria of June27.

Name: D0-6-27-1 Concentration: 3.0 ng/ul A260/280: 2.36

Name: D0-6-27-2 Concentration: 2.0 ng/ul A260/280: -8.61

Name: D0-6-27-3 Concentration: 3.6 ng/ul A260/280: 1.70

Name: D0-6-27-4 Concentration: 3.8 ng/ul A260/280: 1.73

We forget to add ethanol in Solution W2 of Axygen Miniprep Kit, leading the failure of the experiment.

June 28

1. Miniprep plasmid of D0-6-26-2 and pCJDFA from bacteria of June27.

Name: D0-6-28-1 Concentration: 16.2 ng/ul A260/280: 2.11

Name: D0-6-28-2 Concentration: 21.0 ng/ul A260/280: 1.80

[Sequencing]

Name: D0-6-28-3 Concentration: 21.2 ng/ul A260/280: 1.77

[Sequencing]

Name: FA-6-28-1 Concentration: 4.3 ng/ul A260/280: 1.79

Name: FA-6-28-2 Concentration: 4.9 ng/ul A260/280: 2.00

Name: FA-6-28-3 Concentration: 7.3 ng/ul A260/280: 1.64

2. Pick up single colony of plate of D0-6-26-2, pCJDFA (DH5α), pCJDFB (DH5α) of June 26 and amplify them in liquid LB medium for minipreparing plasmids later.

3. Restriction enzyme digestion of D0-6-28-1, D0-6-28-2 and D0-6-28-3 with EcoR1 and Pst1.

It seems that we haven't achieved the correct plasmid but this may be caused by low concentration.

June 29

Amplify pCJDFA (DH5α) from bacteria of June 28.

June 30

1. Miniprep plasmid of pCJDFA (DH5α) from bacteria of June 29.

Name: FA-6-29-1 Concentration: 12.5 ng/ul A260/280: 1.29 [Sequencing]

Name: FA-6-29-2 Concentration: 12.7 ng/ul A260/280: 1.49 [Sequencing]

Name: FA-6-29-3 Concentration: 7.2 ng/ul A260/280: 0.96

2. Amplify pCJDFB (DH5α) and pCJDFB (DH10β) from bacteria of June28.

July 01

1. Receive correct sequencing results of D0-6-28-2 and D0-6-28-3.

2. Amplify bacteria of D0-6-28-2 and D0-6-28-3 for glycerol stock.

3. Miniprep plasmid of pCJDFB from bacteria of June 30.

Name: FB-7-01-1 Concentration: 7.1 ng/ul A260/280: 2.20

Name: FB-7-01-2 Concentration: 25.8 ng/ul A260/280: 2.05

[Sequencing]

Name: FB-7-01-3 Concentration: 20.4 ng/ul A260/280: 2.03

Name: FB-7-01-4 Concentration: 18.0 ng/ul A260/280: 1.83

[Sequencing]

Week 3 (07.02-07.08): Co-transformation of pCJDD0, pCJDFA, pCJDFB

July 02

1. Prepare LB plates of chloramphenicol resistance.

2. Sequencing of FA-6-29-2 and FA-6-29-2 failed.

July 03

1. Amplify pCJDFA (DH5α) from bacteria of June 29 for sequencing.

2. Co-transformation of pCJDD0, pCJDFA, pCJDFB into BL21*(DE3). Besides, transformation of pCJDD0, pCJDFA, pCJDFB into BL21*(DE3) respectively.

July 04

1. Pick up single colony of plate of D0 (BL21*(DE3)) of July 03 and amplify them in liquid LB medium.

2. Pick up single colony of plate of co-transformation of pCJDD0, pCJDFA, pCJDFB (BL21*(DE3)) of July 03 and amplify them on other LB plate.

July 05

1. Amplify co-transformation bacteria of July 04, FB bacteria of July 03 and E.coli BL21*(DE3) strain in liquid LB medium.

2. Make glycerol stock of D0 (BL21*(DE3)).

3. Transform pCJDFA into (BL21*(DE3) for transformation of July 03 failed.

July 06

1. Measure growth curve of D0 (BL21*(DE3)) and BL21*(DE3), respectively. We mistook the absorbance wavelength of spectrophotometer NanoDrop.

2. Pick up a single colony of plate of FA (BL21*(DE3)) of July 03 and amplify it in liquid LB medium.

3. Pick up a single colony of plate of co-transformation of July 04 and amplify it in liquid LB medium.

4. Amplify co-transformation bacteria of July 06 E.coli BL21*(DE3) strain in liquid LB medium for measurement of growth curve.

5. Make glycerol stock of FA (BL21*(DE3)), FB (BL21*(DE3)) and co-transformation (BL21*(DE3)).

6. Amplify backbone pSB1C3 by amplifying part in plate 4, 1E, 3E. Transform them into DH10β.

July 07

1. Miniprep plasmid of pCJDFB from bacteria of July 06.

Name: FB-7-07 Concentration: 14.6 ng/ul A260/280: 1.73

Name: Co-trans-7-07 Concentration: 9.7 ng/ul A260/280: 1.79

2. Make the first part K738000, RNA scaffold with promoter and terminator. Do PCR procedure.

PCR of D0 seems to be correct, but there is nothing in pCJDFB and the plasmid of co-transformation bacteria is the same as that of pCJDD0.

3. Re-transformation:

No. name resistance plate strain

1 co-transform-2-1 A+K+S Sp BL21*DE3

2 co-transform-2-1 A+K+S Kan BL21*DE3

3 orginal FA Sp Sp DH10β

4 orginal FB Kan Kan DH10β

5 FA-6-30-1 Sp Sp DH10β

6 FB-7-01-4 Kan Kan DH10β

7 plate3 16H Kan Kan BL21*DE3

When coated the plates, we made a mistake an put No.3 and No.4 bacteria together on Sp resistance plate, thus somehow leading to failure of these two transformations.

4. PCR purification of D0 PCR product.

5. Inoculate D0 bacteria in 3 resistance liquid LB medium to see if there is problem with antibiotics.

6. Double digest D0-PCR-product and pSB1C3 with EcoR1 and Pst1.

7. One of our teammate went to Peking and communicate with iGEM team there.

July 08

1. Measure growth curve: inoculate co-transformation 2-1 bacteria 1 ml into 50 ml liquid LB medium. Measurement was done with 723-Spectrum. It grew so slow that we gave up again.

2. Test the efficiency of competent cells with part in plate 1, 1G.

3. Amplify FA-7-3-1 and FA-7-3-2 bacteria for minipreparing plasmids later.

4. Miniprep plasmid of pCJDFB from bacteria of July 06.

Name: FA-7-8-1 Concentration: 54.1 ng/ul A260/280: 1.88

Name: FA-7-8-2 Concentration: 45.1 ng/ul A260/280: 1.89

The concentration is the highest among all pCJDFA we have miniprepared by now. However, its electrophoresis result is not correct at all.

5. Amplify glycerol stock bacteria FA (BL21*(DE3)) and FB (BL21*(DE3)) on plates.

6. Purify the products of double digestion of July 07.

Week 4 (07.09-07.15): Make our own biobrick parts

July 09

Prepare liquid SOB medium for co-transformation.

July 10

Amplify glycerol stock bacteria FB (DH5α) on plates.

July 11

1. Identify FA and FB with PCR.

2. Amplify FB-7-10-plate bacteria in liquid LB medium.

July 12

1. Miniprep plasmid of pCJDFB from bacteria of July 06.

Name: FB-7-11 Concentration: 15.4 ng/ul A260/280: 1.88

2. Amplify FB-7-10-plate1, FB-7-10-plate2, FA-6.29-liq (DH5α), FA-lix and FB-lix bacteria for minipreparing plasmids later.

3. Identify FB with PCR again.We found there were problems with primers used in those reactions, thus leading to the failure.

4. Ligation: D0 and pSB1C3, purified at July 08.

July 13

1. Transformation:

No.name time of heat shock/s

1 D0-FA-FB 90

2 FA-FB 90

3 D0-FA-FB 30

4 FA-FB 30

5 ligation product 90

6 D0 90

2. Miniprep plasmid of FA and FB from bacteria of July 12.

Name: FA-7-13-1-1 Concentration: 15.8 ng/ul A260/280: 1.89

Name: FA-7-13-1-2 Concentration: 10.8 ng/ul A260/280: 2.06

Name: FA-7-13-1-3 Concentration: 13.6 ng/ul A260/280: 2.30

Name: FA-7-13-1-4 Concentration: 12.3 ng/ul A260/280: 2.13

Name: FA-7-13-2-1 Concentration: 9.4 ng/ul A260/280: 2.15

Name: FA-7-13-2-2 Concentration: 12.4 ng/ul A260/280: 1.94

Name: FA-7-13-2-3 Concentration: 20.1 ng/ul A260/280: 1.94

Name: FA-7-13-3-1 Concentration: 11.1 ng/ul A260/280: 2.23

Name: FA-7-13-lix Concentration: 13.3 ng/ul A260/280: 2.08

Name: FB-7-13-yy Concentration: 26.4 ng/ul A260/280: 1.84

July 14

1. Co-transformation:

No.name time of heat shock/s

1 D0-FA-FB 90

2 D0-FA-FB 30

Use SOC liquid medium.

2. Miniprep plasmid of FA and FB from bacteria of July 12.

Name: FB-7-14-1 Concentration: 42.9 ng/ul A260/280: 0.67

Name: FB-7-14-2 Concentration: 21.0 ng/ul A260/280: 2.69

Name: FB-7-14-3 Concentration: 44.7 ng/ul A260/280: 0.59

Name: FB-7-14-4 Concentration: 14.4 ng/ul A260/280: 2.09

Name: FB-7-14-5 Concentration: 33.1 ng/ul A260/280: 0.68

Name: FB-7-14-6 Concentration: 26.1 ng/ul A260/280: 1.64

3. Amplify glycerol stock bacteria co-transformation (BL21*(DE3)) on 3 resistance plates, glycerol stock bacteria D0 on Amp plate.

4. Amplify FB-7-12-2, FB-7-12-4, FA-7-12-2-2 and FA-7-12-2-3 in LB liquid medium, named FB-7-14-2, FB-7-14-4, FA-7-14-2-2 and FA-7-14-2-3.

5. Digest FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with Pst1.

Maybe the concentration is too low that we can't recognize it.

July 15

1. Transformation:

No. name time of heat shock/s Strain

1 D0-FA-FB 90 DH10β

2 FA-FB 90 DH10β

3 D0-FA-FB 30 BL21*(DE3)

4 FA-FB 30 BL21*(DE3)

5 D0-pSB1C3 90 BL21*(DE3)

6 D0 90 BL21*(DE3)

2. Amplify bacteria in LB liquid medium:

No. Name From quantity

1 Co-7-15 co-transformation (BL21*(DE3)) 6

2 D0-7-15 D0-7-14 1

3 Co3-7-15-1-3-1 Co3-7-14-1 1

4 Co3-7-15-1-3-2 Co3-7-14-2 1

Week 5 (07.16-07.22): Struggle to acquire correct plasmids

July 16

1. Miniprep plasmid of FA and FB from bacteria of July 12.

Name: D0-7-16-1 Concentration: 60.9 ng/ul A260/280: 1.65

Name: D0-7-16-2 Concentration: 34.0 ng/ul A260/280: 1.93

Name: Penta Concentration: 74.9 ng/ul A260/280: 1.91

Name: RFP Concentration: 201.2 ng/ul A260/280: 1.90

2. Amplify glycerol stock D0 bacteria on two Amp plates, named D0-7-16-1 and D0-7-16-2.

3. Identify FA-7-13-2-2, FA-7-13-2-3, FB-7-14-2 and FB-7-14-4 with PCR, new primers.

The result of FA is not very ideal, while that of FB is not very exactly. The plasmid (all was Miniprep today) seems just stay where they were put in.

July 17

1. Amplify FA-FB-7-15-90 and FA-FB-7-15-30 in LB liquid medium.

2. Identify FA-7-13-2-2, FA-7-13-3-1 with PCR, increase Tm by 2Degrees Celsius.

3. Amplify glycerol stock co-transformation-2-1 bacteria on plate, named co3-7-17.

July 18

Amplify FA-7-13-2-2, FA-7-13-2-3, FB-7-13-2 and FB-7-13-5 for minipreparing plasmids later.

July 19

1. Co-transformation of D0-7-16-2, FA-7-13-1-1 and FB-7-14-6, named Co3-7-18.

2. Amplify FB-7-12-1 and FB-7-12-3 in LB liquid medium.

3. Miniprep plasmid of FA-7-18-2-2 and FA-7-18-2-3.

Name: FA-7-18-2-2 Concentration: 17.4 ng/ul A260/280: 2.19

Name: FA-7-18-2-3 Concentration: 14.7 ng/ul A260/280: 2.27

July 20

Amplify glycerol stock Co3-2-1 bacteria on plate, named Co3-7-20.

July 21

1. Gradient PCR to find an appropriate Tm value for FA.

It seems that there is not a lot of differences.

2. Amplify Co3-7-20 on plates, named Co3-7-21.

3. Amplify the following bacteria:

No Name Form resistance plate

1 Co2-7-21-1 Co2-7-17-1 K+S K+S

2 Co2-7-21-2 Co2-7-17-2 K+S K+S

3 Co2-7-21-3 Co2-7-17-3 K+S K+S

4 control-kan DH10β - Kan

5 control-sp DH10β - Sp

6 control DH10β - -

July 22

1. Amplify Co3-7-21 in LB liquid medium, named Co3-7-22.

2. Colony PCR to test Co2-7-21, Co3-7-22, and FB-7-19.

We found that we may use a wrong protocol of colony PCR.

3. Co-transform D0, FA and FB, named Co3-7-22. Co-transform FA and FB, named Co2-7-22.

4. Amplify FA-7-18-2-2, FA-7-18-2-3, FB-7-18-2 for minipreparing plasmids later.

5. Amplify D0-7-15-2 and Co3-7-21 on other plate, named D0-7-22-2 and Co3-7-22.

Week 6 (07.23-07.29): Re-sequencing and re-test our original plasmids

July 23

1. Amplify bacteria of FA-7-22 for glycerol stock.

2. Miniprep plasmid of FA-7-22-2-2 and FA-7-22-2-3.

Name: FA-7-22-2-2-1 Concentration: 20.1 ng/ul A260/280: 2.19

Name: FA-7-22-2-2-2 Concentration: 15.3 ng/ul A260/280: 2.05

Name: FA-7-22-2-3-1 Concentration: 24.2 ng/ul A260/280: 1.91

Name: FA-7-22-2-3-2 Concentration: 22.3 ng/ul A260/280: 1.69

3. Amplify glycerol stock bacteria FB on plates, named FB-7-23.

4. Amplify DH10β on plate.

5. Amplify D0-7-23 plate in LB liquid medium.

6. Colony PCR of FA, FB and Co3.

7. Miniprep plasmid of Co3-7-22-1 and Co3-7-22-2 for PCR procedure.

Name: Co3-7-23-1 Concentration: 29.9 ng/ul A260/280: 1.91

Name: Co3-7-23-2 Concentration: 18.9 ng/ul A260/280: 2.00

8. Amplify Co3-7-22-1, Co3-7-22-2, Co2-7-17-1, Co2-7-17-2 and Co2-7-17-3, named Co3-7-23-1, Co3-7-23-2, Co2-7-23-1, Co2-7-23-2 and Co2-7-23-3.

July 24

1. Miniprep plasmid of D0-7-23 and Co3-7-22-2 for PCR procedure.

Name: D0-7-24-2-1 Concentration: 40.6 ng/ul A260/280: 2.00

Name: D0-7-24-2-2 Concentration: 43.7 ng/ul A260/280: 1.98

Name: D0-7-24-2-3 Concentration: 42.3 ng/ul A260/280: 2.03

Name: D0-7-24-2-4 Concentration: 51.7 ng/ul A260/280: 1.87

Name: D0-7-24-2-5 Concentration: 40.7 ng/ul A260/280: 1.87

Name: D0-7-24-2-6 Concentration: 28.9 ng/ul A260/280: 1.83

Name: FB-7-24-2-1 Concentration: 18.2 ng/ul A260/280: 2.32

Name: FB-7-24-2-4 Concentration: 16.6 ng/ul A260/280: 1.95

Name: Co-7-24-3 Concentration: 27.1 ng/ul A260/280: 1.84

Name: Co-7-24-9 Concentration: 15.3 ng/ul A260/280: 1.81

Name: Co-7-24-10 Concentration: 27.0 ng/ul A260/280: 1.78

Name: Co-7-24-12 Concentration: 15.5 ng/ul A260/280: 1.77

2. Amplify FB-7-22-2 and FB-7-23 on other plates, named FB-7-24-2 and FB-7-24.

3. Transform FA-7-23-2-3-1 and FB-7-24-2-4 into BL21*(DE3), named Co2-7-24.

4. PCR to test (1) if pCJDD0 reacts with primer FAF and FAR; (2) further confirm the success of co-transformation.

Unfortunately, we found we can achieve the same result of PCR whatever the template is if we use the same pare of primers. In contrast, we can't find there is any similarity between template and primers. We are really confused about that. It means that we can't test the success of co-transformation by PCR.

5. Amplify FB-7-23 bacteria in LB liquid medium.

July 25

Amplify Co-7-24-1-1, Co-7-24-1-2, Co-7-24-2-1 and Co-7-24-2-2, named Co-7-25-1-1, Co-7-25-1-2, Co-7-25-2-1 and Co-7-25-2-2. We prepared to induce them with IPTG later.

July 26

Transform original plasmids pCJDFA, pCJDFB and pCJDD0 into DH5α competent cells.

July 27

Peking iGEM_12 team visited us, we had a dinner together.

July 28

1. Peking and we ZJU introduced our projects this year briefly.

2. Amplify BL21(DE3)plyss strain on LB plates.

3. Transform FA-7-27-1, FA-7-27-2, FB-7-27-1 and FB-7-27-2 into DH5α competent cells respectively, named FA-7-28-1, FA-7-28-2, FB-7-28-1 and FB-7-28-2.

4. Ligation: D0 and pSB1C3, purified at July 08, named the product as K738000. Incubate at 22Degrees Celsius for 1h to increase the efficiency of transformation later.

5. Transform K738000 into DH5α competent cells, named K738000-7-28.

July 29

1. Amplify bacteria BL21(DE3)plyss in LB liquid medium for glycerol stock.

2. Amplify K738000-7-28 in LB liquid medium, named K738000-7-29.

Week 7 (07.30-08.05): Test part BBa_K537009 and make our own parts

July 30

1. Miniprep plasmid of K738000-7-29.

Name: K738000-7-30-1 Concentration: 60.0 ng/ul A260/280: 1.86

Name: K738000-7-30-2 Concentration: 47.1 ng/ul A260/280: 1.95

Name: K738000-7-30-3 Concentration: 90.1 ng/ul A260/280: 1.95

Name: K738000-7-30-10 Concentration: 43.6 ng/ul A260/280: 1.98

PCR of K738000-7-30

Unfortunately, there was a slight electrophoretic band in control. But the sequencing result came later verified that we've got the correct ligation product.

2. Pick up single colonies from plate of FB-7-26-2 and amplify it in LB liquid medium.

July 31

1. Amplify backbone pSB1C3:

Double digest K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.

2. Miniprep plasmid of K738000-7-29 and FA-7-28-1.

Name: K738000-7-30-4 Concentration: 83.9 ng/ul A260/280: 1.94

Name: K738000-7-30-5 Concentration: 50.1 ng/ul A260/280: 2.00

Name: K738000-7-30-11 Concentration: 46.0 ng/ul A260/280: 1.97

Name: FA-7-28-1 Concentration: 33.0 ng/ul A260/280: 2.04

August 01

Double digest K738000-7-23-2 and K738000-7-23-3 with EcoR1 and Pst1 and got linear backbone through DNA gel extraction.

August 02

1. Transform part K537009 into DH5α to acquire experience, named venus-8-2.

2. Transform original pCJDFA and pCJDFB into DH5α, named FA-8-2 and FB-8-2.

3. Transform part K398326 into DH10β to amplify backbone pSB1C3 easier, BB-8-2.

August 03

1. Amplify BB-8-2, FA-8-2 and FB -8-2 for minipreparing plasmids later, named BB-8-3, FA-8-3 and FB-8-3.

August 04

1. Miniprep plasmid of BB-8-3 FA-8-3 and FB-8-3.

Name: BB-8-4 Concentration: 76.2 ng/ul A260/280: 1.91

Name: BB-8-4 Concentration: 40.3 ng/ul A260/280: 1.90

Name: FA-8-4 Concentration: 19.6 ng/ul A260/280: 1.97

Name: FB-8-4 Concentration: 35.9 ng/ul A260/280: 1.98

Unfortunately, the PCR results were so different from that of original plasmids. We couldn't figure out the reason.

2. Amplify Venus-8-2 in LB liquid medium, named Venus-8-4.

3. Transform FAM-8-4-1, FAM-8-4-1, FBP-8-4-1 and FBP-8-4-1 into DH5α.

August 05

1. Pick up single colonies from plate of FAM-8-4 and FBP-8-4 and amplify it in LB liquid medium and LB plates.

2. Colony PCR of FAM-8-4-2 and FBP-8-4-2

3. Double digest BB-8-4-1 with (1) EcoR1 and Pst1, (2) Xba1 and Spe1, get linear backbone through PCR cleanup kit.

4. Miniprep plasmid of Venus-8-4.

Name: Venus-8-5-1 Concentration: 168.1 ng/ul A260/280: 1.91

Name: Venus-8-5-1 Concentration: 83.4 ng/ul A260/280: 1.93

5. Pick up single colonies from plate of FAM-8-4-1, FAM-8-4-2, FBP-8-4-1 and FBP-8-4-2 and amplify it in LB liquid medium and LB plates. Miniprep plasmid of them.

Name: FAM-8-5-1 Concentration: 69.9 ng/ul A260/280: 1.95

Name: FAM-8-5-2 Concentration: 57.3 ng/ul A260/280: 1.95

Name: FAM-8-5-3 Concentration: 86.8 ng/ul A260/280: 2.06

Name: FBP-8-5-2 Concentration: 21.0 ng/ul A260/280: 2.14

Name: FBP-8-5-3 Concentration: 15.5 ng/ul A260/280: 2.14

Name: FBP-8-5-5 Concentration: 15.6 ng/ul A260/280: 2.07

6. Co-transform FA-8-5-3, FB-8-5-2 and D0-4 into BL21*(DE3). Co-transform FA-8-5-3 and FB-8-5-2 into BL21*(DE3).

Week 8 (08.06-08.12):

August 06

1. Amplify Venus-8-2 in LB liquid medium. Add 0mM, 0.5mM, 1mM, 1.5mM theophylline accordingly.

2. PCR of part K738001:

3. PCR of original pCJDFA and pCJDFB.

4. Amplify FAM-8-5 and FBP-8-5 for minipreparing plasmids.

Name: FAM-8-6-1 Concentration: 56.7 ng/ul A260/280: 1.95

Name: FAM-8-6-2 Concentration: 57.3 ng/ul A260/280: 1.95

Name: FAM-8-6-3 Concentration: 86.8 ng/ul A260/280: 2.06

Name: FBP-8-6-1 Concentration: 21.0 ng/ul A260/280: 2.14

Name: FBP-8-6-2 Concentration: 15.5 ng/ul A260/280: 2.14

Name: FBP-8-6-3 Concentration: 15.6 ng/ul A260/280: 2.07

5. Add theophylline 0.08g/10mL into Venus-8-6, observe through fluorescence microscope.

6. Purify PCR product of K738001, named K738001-8-6.

7. Co-transform FA-8-5-3, FB-8-5-2 and D0-4 into BL21*(DE3), named Co3-8-6. Co-transform FA-8-5-3 and FB-8-5-2 into BL21*(DE3), named Co2-8-6.

August 07

1. Double digest K738001-8-6 with Xba1 and Spe1. Purify the product before ligation step.

2. Ligation: K738001-8-6 and pSB1C3.

3. Transform the product of ligation into DH5α competent cells.

August 08

Pick up single colonies from plate of K738001-8-7 and amplify it in LB liquid medium and LB plate.

August 09

1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium, induce with IPTG when OD600 is about 0.6 (mid log stage).

2. Miniprep plasmids of K738001-8-8:

Name: K738001-8-9-1 Concentration: 174.2 ng/ul A260/280: 1.91

Name: K738001-8-9-2 Concentration: 298.5 ng/ul A260/280: 1.87

3. Amplify FBP overnight for minipreparing plasmids later.

4. Mutation of D0:

PCR1: delete 3bp after MS2 in D0.

PCR2: delete 3bp before MS2 in D0.

August 10

1. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Take photos with fluorescence microscope.

2. Miniprep plasmids of FB-8-9.

Name: FB-8-10-1 Concentration: 14.6 ng/ul A260/280: 1.97

Name: FB-8-10-2 Concentration: 24.4 ng/ul A260/280: 1.93

3. Mutation of D0:

PCR1: PCR with primer P1 and P2. Set a temperature gradient from 44Degrees Celsius to 54Degrees Celsius.

PCR2: product of 47.5Degrees Celsius from PCR1 20 ul + Taq Mix 37.5 ul + D0RLX 1.5 ul + ddH2O 36 ul.

PCR3: 49.6Degrees Celsius product 20 ul + 10 ul Taq Mix +0.5 ul D0RLX + 9.5 ul ddH2O.

4. Amplify FB-8-7 in LB liquid medium.

August 11

1. Miniprep plasmids of FB-8-11.

Name: FB-8-11-1 Concentration: 33.9 ng/ul A260/280: 2.06

Name: FB-8-11-2 Concentration: 50.2 ng/ul A260/280: 2.04

2. Amplify Co2-8-7 and Co3-8-7 in LB liquid medium. Induce with IPTG when OD600 is about 0.6 (mid log stage). Test fluorescence strength with hybrid synergy reader. Exciting light 535nm, absorbing light 480nm.

August 12

Week 9 bogged, Subversion and start again

2012/8/13 Monday, August 13, 2012

Yu Jianing and Yan came to Prof. Chen Xin for MD (molecular dynamics simulation, suggested by PKU iGem) modeling in RNA scaffold project. Prof. CHEN pointed out that it's so hard and costing for us green hand undergraduates to manipulate MD, which could be replaced by RNA prediction software and ODE modeling with matlab.

Zhang and Chen transform a group of D0, FA and FB with different combination.

LIU Xiao comes to carry out point mutation experiment to modify origin D0 in PCR method. He intended to build a library. However, we have only one month left, too short to finish the library.

Yan's fluorescent test with part K537009 is modestly successful. Most excitingly, Yan and Chen capture a love heart (or you can refer to it as a smiling smile) imaging by E.coli.

2012/8/14, Tuesday 14, 2012

Chen makes competent cell with BL21 star DE3 borrow from Zhejiang Sci-Tech University.

2012/8/15 Wednesday, August 15, 2012

Prof. SHAO Jianzhong is willing to let us use the Synergy hybrid reader in his lab.

Chen and Zhang test fluorescence of D0, FA,FB,FA+D0,FA+FB,FA+FB+D0 with IPTG concentration gradient but find out that the fluorescence intensity are nearly no difference comparing with scaffold and without scaffold surprisingly.

2012/8/16 Thursday, August 16, 2012

After senior fellow's advises, however, we still can't see the result in < Organization of Intracellular Reactions with Rationally Designed RNA assemblies>.

Yan tried the parts of Theophylline riboswitch with YFP Venus reporter and it worked. (Though excite 515nm emit 528 mn is too close for Synergy hybrid reader to detect and we change it into 505nm and 535nm.)

You can see the fluorescence intensity increasing with Theophylline concentration.

All night talks and strange Sequencing results make us skeptical about the FAFB plasmids sent by French lab (which is crucial to our project). Maybe there's something wrong with our raw material.

2012/8/17 Friday, August 17, 2012

Today we faced mega-disaster that most of us thought the plasmids: FA & FB sent early August are not what we were asking for. Dr. Delebecque is so helpful and patient but he's away from his French lab now. He sent us an email providing a lot of information we need if we construct the plasmid on our own. So we have to go back and start from the very beginning. Chen, Zhang and Liu try to redesign the primers; Prof. Shao lent us the eGFP. Our advisor, Li Xin comes to discuss with us the technology of construct.

"You must be ready to burn yourself in your own flame: how could you become new, if you had not first become ashes?" --When Nietzsche Wept

2012/8/18 Saturday, August 18, 2012

1 We bought commercial plasmids pCOLADuet?-1 Vector and pCDFDuet-1 Vector, and streak plate them respectively in Spectinomycin, Kanamycin and LB plate.

2 The part "2012 plate 1 15P" can't be transformed, as Zhang and Yan tried it twice.

2012/8/19 Sunday, August 19, 2012

1 plasmid miniprep:

Plasmid Concentration A260/280 A260/230

pCOLADuet?-1(1) 29.9 ng/μL 1.96 1.21

pCOLADuet?-1(2) 25.6 ng/μL 2.07 1.19

pCOLADuet?-1(3) 28.0 ng/μL 2.07 1.15

2 LIU Xiao continues his point mutation experiment to modify origin D0 in PCR and build a library of changed RNA scaffold with different aptamer arm length.

He makes a new RNA scaffold, Nucleic Acid Electrophoresis as follows.

First step:

Second step:

First step product is due in 50bp and second is due in 200bp. But the Electrophoresis effect is not good.

3. Freshmen come to us and we taught them some methods for using Library Resources and making it in college.

Week 10 keep moving on

2012/8/20 Monday, August 20, 2012

1. Zhang continues LIU Xiao's point mutation D0 library experiment. According to the instructions of DNA gel extraction kit, she recover enzyme-digested products product pETDuet-BB, put new D0 into it.

2. We receive part K411003 and plate it on Chloramphenicol plates, and incubate.

2012/8/21 Tuesday, August 21, 2012

1. K411003 (Taipei 2010) were adding a series of Theophylline. This experiment is used both for testing parts and fishing for the condition of our synthetic D0 from Genescript's future GFP experiment.

2. The Synergy hybrid reader data is good. You can see the fluorescence intensity increasing with Theophylline concentration.

2012/8/22 Wednesday, August 22, 2012

1. 00:39 in the morning, YU Jianing sends an e-mail to Yan from her hometown, attaching version 1 ODE modeling and some questions about biology meanings.

2. We start our major experiment: construct FA-2X-MS2 and FB-2X-PP7

2.1 Fragment

1. We PCR FA and FB from pEGFP.

2. We PCR MS2 from FAM( from Dr. Delebecque)

3. We PCR PP7 from FBP(from Dr. Delebecque )

Then use gel extraction kit to get these fragment.

2.2 Overlap PCR

First overlap PCR in our lab. We uses precisely designed primers to make FA and MS2 linked with 2X. So does FB and PP7.

3. Zhang goes on with LIU Xiao's point mutation and use Megaprimer PCR methods.

2012/8/23 Thursday, August 23, 2012

1. Yan PCR riboscaffold (synthesis by Genescript) and make it into Part.

2. Zhang continues mutation D0 and enzyme digest the fragment for PCR test.

3. LIU Huachun uses some parts form registry to construct a new EGFP for positive control.

2012/8/24 Friday, August 24, 2012

1. LIU reproduce pSB1C3

2. Yan continues to make part of riboscaffold.

3. Chen use Taq DNA Polymerase (ordinary) to put A onto FA-2X-MS2 and FB-2X-PP7 and link it onto T vector.

2012/8/25 Saturday, August 25, 2012

1. We extracted and amplified the mutation D0 library and then sent it to a sequencing facility

2. LIU's pCDF-EGFP: transform into DH5α (It works pretty well.)

3. Miniprep: Yan's last day's part riboscaffold

2012/8/26 Sunday, August 26, 2012

1. Miniprep: pCDFDuet-EGFP and pColaDuet

2. Part riboscaffold PCR: a little different from its original PCR from pETDuet Genescript. So we should send sequencing for validation.

Week 11 Recuperate

2012/8/27 Monday, August 27, 2012

1. Chen did the overlap PCR and construct FB-2X-PP7 (approximately 630 bp). It seems correct in the agarose gel electrophoresis. (1000 marker on the left and 5000 marker on the right)

2. Yan and Chen PCR MS2 from plasmids Dr. Delebecque sent us respectively. Yan uses 57.5 Degrees Celsius as melting temperature and Chen uses 58.5 and 63.5. But the electrophoretic bands seem incorrect. (Next day we find angrily that the primer the company synthesized is wrong!)

2012/8/28 Tuesday, August 28, 2012

1. LIU uses gel extraction to get last day Chen's MS2 and carry on overlap PCR to make FA-2X-MS2.

2. Chen transforms FB-2X-PP7 (T vector) into Top 10.

3. Repeated tests: Yan does the same as LIU and sets annealing temperature 59Degrees Celsius and 61Degrees Celsius.

2012/8/29 Wednesday, August 29, 2012

4. Teacher Hu Yuhua from Edinburgh University interviews us concerning team collaboration. She offers that if we have oral English barriers we could come to her.

5. LIU makes competent cell of DH10β

2012/8/30 Thursday, August 30, 2012

All of us attend short semester and did nothing about igem today.

2012/8/31 Friday, August 31, 2012

1. Sequencing result shows that FB-2X-PP7 we have constructed was correct in T vector. So LIU prepare some for Miniprep and glycerol freeze store.

2. Enzyme digestion and ligation: make FB-2X-PP7 from T vector into pColaDuet and transform it in DH5α.