Team:OUC-China/Notebook

• Week 1 (April21 – April 26)

Wet Lab Overview

To achieve the expected results we discussed and proposed four plans: A) Free ferritins combined with RNA thermometer. B) MTS (Membrane Targeting Sequence)-Ferritins combined with Two Component System. C) Free ferritins combined with free inteins. D) A fusion of ferritin subunit and intein. For Mag-receiver and Thermo-regulator, we chose to construct ferritin ftnA and RNA thermometer first.

Mag-receiver

To obtain ftnA gene by PCR, Top 10 cells were cultured in LB liquid medium. After failing in PCR for several days, Jinyang Liang and Qikai Qin decided to extract genome DNA from Top 10 instead of doing PCR directly.

Fig.1. Lane 7 is DL 2000 marker. Lane 1-6 are PCR peoducts(ftnA) cloned from Top 10, used different concentrations of culture. The Agarose gel electrophoresis shows bands between 500bpand750bp, the target sequence band is expected at 500bp. The sequencing results validated the correctness of the PCR products.

Qikai Qin tried to assemble ftnA into T-vector, then Cun Wei and Renjie Shang transformed the plasmids into Top 10 cells to amplificate ftnA gene. It’s a pity that there were false positive phenomenon when using blue-white screen.

Thermo-regulator

The experiments were designed to test the performance of three alternative RNA thermometers.

Dry Lab Overview

(Theory Part)After setting our four plans, we started to search for references about ferritin and nanoparticles. We wanted to find a specific method to calculate the heating power of the ferritins and adjust it into our project.
(Device Part)For the need of electromagnetic field, we decided to make a device to generate it.

• Week 2 (April 27 – May 3)

Wet Lab Overview

This week we decided to insert ftnA gene into plasmid pET-28a, because pET-28a is a high expression plasmid. We found that the Amp was ineffective, that’s why the transformation of T-vector was failed. The ftnA gene was inserted into T-vector because double digest experiment of PCR products was unsatisfactory.
We started constructing RNA thermometer circuits.

Mag-receiver

Qikai Qin extracted plasmid pET-28a.
Qikai Qin and Jinyang Liang repeated the PCR, digested the pET-28a and PCR products (ftnA) with BamHI and HindIII-HF, assembled the enzyme-digested products, and transformed the plasmids. PCR identification proved that the experiment failed. We thought that we should purified the enzyme-digested products before ligation.
Jinyang Liang extracted plasmid pET-28a.
After finding the Amp was ineffective, Cun Wei and JieLian repeated the ligation of ftnA and T-vector and transformation. Blue colony appeared on the plate. Renjie Shang and Jinyang Liang inoculated white colonies into LB liquid medium for digestion to identify the ligation.

Fig.2. Digestion of T-Vector-ftnA. The Agarose gel electrophoresis shows bands between 500bpand750bp, the target sequence band is expected at 500bp.

Strains containing T-vector-ftnA were preserved. Jinyang Liang extracted plasmid T-vector-ftnA, digested T-vector-ftnA and pET-28a with BamHI and HindIII-HF. The Agarose gel electrophoresis shows the digest result is unsatisfactory.
Agarose gel electrophoresis was done to test the correctness of plasmid T-ftnA.
Jinyang Liang and Qikai Qin did gel extraction of enzyme digest products, the agarose gel shows the double enzyme digest experiment result is terrible.

Thermo-regulator

Parts J23119 (J), K115001 (K1), K115002 (K2), K115003 (K3), E1010 (E) were got from 2012, 2014 and 2015 kits, and were transformed into E. coli Top 10 strains which were spread on LB ager culture medium with relevant antibiotic later. The sequencing results match the correct one.
Qikai Qin and Zhuo Pan extracted plasmids E1010, J23119, K115001, K115002, and K115003. Cun Wei digested K1, K2, K3 with Xbal and PstI, digested J with SpeI and PstI. Ligation of J with K1, K2, and K3 respectively was down by Cun Wei after gel extraction and agarose gel electrophoresis, then they were transformed into Top 10 cells. There are no colonies growing on the plate.
Cun Wei did gel extraction of E and K1, K2, K3.Zhuo Pan digested K1, K2, K3 with SpeI and PstI, digested E with XbaI and PstI. Ligation of E with K1, K2, and K3 respectively was down by Cun Wei after Gel extraction and Agarose gel electrophoresis, then they were transformed into Top 10 cells. There are no colonies growing on the plate.

Dry Lab Overview

(Theory Part)Ruipeng Wei started from ftnA first to calculate the heating power and efficiency.
(Device Part)For the specific frequency and magnetic strength we need, Weiyan Chen chose the Seiler Oscillation circuit to be the basic generator circuit.

• Week 3 (May 4 – May10)

Wet Lab Overview

This week, we proposed three plans for mag-receiver: A) Insert H-L gene into pET28a or pSB1C3. B) Insert H-linker-L gene into pET28a or pSB1C3. C) Insert ftnA gene into pET28a.
The low efficiency of double enzyme digest puzzled us.

Mag-receiver

JieLian and Qingdian Mu extracted plasmid T-ftnA. Jinyang Liang extracted plasmid pET-28a and purified both two plasmids. Qingdian Mu and Jinyang Liang digested plasmids with BamHI and HindIII-HF. JieLian and Qingdian Mu conducted experiments of double enzyme digest and single enzyme digest, finding that there are subtle bands at 500bp for double enzyme digest.
JieLian and Jinyang Liang successfully digested purified plasmids with BamHI and HindIII-HF at the second attempt.

Fig.3. Lane 1 and 10 are DL 5000 marker. Lane 2-4 are pET28a digested by BamHI and HindIII, used different concentratioms of culture. Lane 5-7 are their control group.

Jinyang Liang extracted pET-28a, JieLian and Qingdian Mu digested pET-28a successfully. Jinyang Liang purified enzyme digested products, assembled and transformed pET-ftnA into BL21 (DE3) competent cells.

Thermo-regulator

Cun Wei digested K1, K2, K3 with SpeI and PstI, digested E with XbaI and PstI. Gel extraction was down and then Cun Wei did agarose gel electrophoresis to estimate the concentration of target bands. Ligation of E and K1, E and K2, E and E3 were respectively down by Cun Wei. The ligation products K1E, K2E, K3E were transformed into Top 10 cells by Zhuo Pan. There are no colonies growing on the plate.
Cun Wei and Zhuo Pan extracted a lot of plasmids loaded partsJ23119 (J), K115001 (K1), K115002 (K2), K115003 (K3), E1010 (E). Cun Wei did agarose gel electrophoresis to test the products of plasmid extraction and then abandoned some of them. Zhuo pan digested E with XbaI and PstI, digested K1, K2, K3 with SpeI and PstI. Gel extraction was down by Cun Wei. Agarose gel electrophoresis was down to estimate the concentration of target bands. Ligation and transformation were down by Cun Wei and Zhuo Pan. There are no colonies growing on the plate.
Cun Wei repeated agarose gel electrophoresis and ligation for the other products of digestion. Transformation was down by Zhuo Pan. There are colonies on the plate finally, some of colonies were inoculated to LB liquid medium for PCR identification and plasmids extraction.

Dry Lab Overview

(Theory Part)After parameter setting and several formula changes, Rupeng Wei finally got a theoretical heating power of FtnA and observed its fluctuation with the variance of MF frequency and strength. After the heating power calculated, we started to observe the ftnA’s expression level and stability.

Fig.4. heating power’s fluctuation with the variance of MF frequency and strength.

(Device Part)Using the simulation software named Multisim, Weiyan Chen optimized the circuit design and improved its stability and feasibility.



• Week 4 (May11 – May17)

Wet Lab Overview

This week we designed the sequence of membrane targeting-ferritin and designed the T7 RNA polymerase and intein as thermo-regulator.
This week we successfully assembled the K115001 with E1010, K115002 with E1010, and K115003 with E1010. There are two reasons why we failed for previous experiments: a) we used buffer as assemble because of the wrong tag. b) The ligation system was incorrect, we summarized that the appropriate proportion of backbone and part is 10:1.

Mag-receiver

JieLian and Qingdian Mu continued constructing pET-28a in case that the first batch was false positive.
Renjie Shang and Jinyang Liang did PCR to identificate the correctness of pET-ftnA.

Fig.5. Lane 11 is DL2000 marker. Lane 1-10 are PCR products cloned from BL21(DE3) monoclonal colony that were transformed pET-28a with ftnA. The Agarose gel electrophoresis shows bands at 800bp, the target sequence band is expected at 794bp.

The same work was repeated for preparing sequencing.

Thermo-regulator

In case of the sequencing results is wrong, the ligation of E and K1 wasn’t stopped. Cun Wei and Zhuo Pan extracted J, K1, K2, K3, E and EK1. Zhuo Pan digested J, K1, K2 and K3 with SpeI and PstI, digested E and K1E with XbaI and SpeI. Ligations of J and K1E, E and K1, K2, K3 were down by Cun Wei after gel extraction and agarose gel electrophoresis. Transformation was down by Zhuo Pan. Only K3E grew on the plate and the PCR identification shows that the ligation of E and K1 last week is failed. Cun Wei did PCR identification of K3E, there are no target bands on the gel.
Cun Wei and Zhuo Pan did gel extraction of K1, K3, J and E which had been digested. Cun Wei and Zhuo Pan did two ligations respectively. Both transformations were failed.
We found the problem of assemble, and ordered new T4 assemble. Cun Wei and Zhuo Pan did four groups of transformation: a) competent cells was spread on the plate without antibiotic. b) Plasmid was transformed into competent cells. c) Product of ligation was transformed into competent cells. d) The product of digestion was transformed into competent cells. Bacterium of a), b) and c) grew on the plate. This shows that the double enzyme digest and transformation has no problems.
Cun Wei and Zhuo Pan digested a lot of K1, K2, K3 and E. Ligation was down after gel extraction and agarose gel electrophoresis. Cun Wei and Zhuo Pan transformed product of ligation into DH5α competent cells. Bacterium of all experimental groups grew on the plate. The PCR identification shows that the ligations of K1 and E, K2 and E, K3 and E are successful.

Fig.6. The agarose gel electrophoresis shows that there are target bands on the gel, the target sequence band K1E is expected around 1040 bp, K2E is expected around 996 bp, K3E is expected around 1053 bp.

Dry Lab Overview

(Theory Part)Ruipeng Wei started to establish the deterministic model with ODEs based on Hill function to describe the expression level of ferritin. Then Ruipeng Wei and Weiyan Chen searched for several references to find out the most optimized parameters and did simulation the first time.
(Device Part)Weiyan Chen and Ruipeng Wei bought all the electronic components for need, and constructed the first electronic plate.



• Week 5 (May 18 – May 24)

Wet Lab Overview

This week we learned SDS PAGE at Professor Chenguang Liu‘s laboratory. We abandon the project of TCS.

Mag-receiver

The sequencing proved the correctness of pET-ftnA.
This week we prepared and tested lab instrument about expressing protein and SDS PAGE.
Jingyang Liang cultured BL21 cells containing pET-ftnA, induced and biomineralized ferritin simultaneously. The strains were prepared for SDS PAGE.
Jinyang Liang harvested the biomineralized strains, resuspended them with PBS, and broke up cells by ultrasound. SDS PAGE was down to identify the ferritin expression in supernatant and sediment. The results shows that the bands stand for sediment is more wider than supernatant, that is, ferritins stayed most in sediments after breaking up cells.

Thermo-regulator

The sample was sent to sequencing, the results match the correct sequences except for K2E.
Cun Wei repeated ligation of K2E.
Cun Wei extracted K1E, K2E, and K3E and did gel extraction of these three plasmids. Zhuo Pan digested K1E, K2E, K3E with XbaI and PstI. Cun Wei did gel extraction after digestion. Ligations of J and K1E, K2E, K3E were down by Cun Wei after gel extraction and agarose gel electrophoresis. Cun We transformed product of ligation into DH5α competent cells. Then Cun Wei send JK1E, JK2E and JK3E to sequencing, the results didn’t match the correct sequences.

Dry Lab Overview

(Theory Part)With the result from ferritin’s deterministic model’s the first simulation, we got an approximate amount of ftnA molecules in the steady state. Then Ruipeng Wei started to establish the deterministic model with ODEs based on mass-law and Michaelis-Menten equations to describe the mechanism of T7 platform.
(Device Part)Weiyan Chen tested the circuit built last week, it worked! So we turned to build the amplifier which can apply the sine signal onto the solenoid. So Weiyan Chen designed the amplifier circuit and simulated the expected effect on the software.

• Week 6 (May25– May 31)

We improved the project of T7. We decided to using ferritin in vivo. We begin to introduce Gibson assembly to our lab.

Wet Lab Overview

We received parts BBa_K1438000, BBa_K1438001, BBa_K1438025, BBa_K1438022, BBa_K1438028, BBa_K1438031, and plasmid pQE-80L from Berlin iGEM team. However, we failed to get the correct and sequenced plasmid, and we had to construct pQE-ferritin by ourselves for future work.

Mag-receiver

Jinyang Liang harvested the biomineralized strains, resuspended them with PBS, and broke up cells by ultrasound.
We first did the agarose gel electrophoresis of Berlin’s plasmids, there are no bands on the gel. Then we did PCR identification of bfr, the products contained the target gene. We transformed Berlin‘s parts into Top 10 competent cells. Jinyang Liang did PCR identification of Berlin‘s parts.

Thermo-regulator

Cun Wei still ligated JK1E, JK2E and JK3E.Cun Wei extracted K1E, K2E, K3E and J plasmids. Then He digested K1E, K2E, K3E with XbaI and PstI and digested J with EcoRI and SpeI. Ligations of J and K1E, K2E, K3E were down by Cun Wei after gel extraction and agarose gel electrophoresis Cun Wei did gel extraction after digestion andtransformed product of ligation into DH5α competent cells. There no target bands on the gel.
Zhuo Pan was responsible for the Gibson assembly. In this week, he consulted some papers and data about Gibson assembly and prepared materials to make the Gibson assembly Mix.

Dry Lab Overview

(Theory Part)For the two deterministic models we did before, parameters finding and adjusting were performed to make the models more rigorous. And Ruipeng Wei did sensitivity analysis to find out which parameter hold the greatest importance in the model of T7 platform and recognized how the intein’s splicing efficiency influences the amount of reporter protein.
(Device Part)We were struck by a serious problem that we need to provide a lot of energy that cannot be afforded by such a simple amplifier to amplify the current strength. So we needed to get another way out. And it took time to search for supports and recalculate.

• Week 7 (June 1 –June7)

Wet Lab Overview

pET-Ecoil-HL was under construction. The big plasmid project and double plasmids project for T7 RNAP were being designed. Gibson assembly was being prepared. We decided to take part in InterLab Study this week. Qikai Qin tried to construct circuits of the three required devices, which are J23108+I13504, J23109+I13504, and J23111+I13504.Two differentpromoter (BBa_J23101, BBa_J23106) was ligated with K1E, K2E and K3E.

Mag-receiver

JieLian did PCR identification of K1189037, there are only primer-dimers on the gel.
Jinyang Liang did PCR again, the Agarose gel electrophoresis shows bands between 1800bp and 2000bp, the target sequence band is expected at 1686bp. The sequencing proved the correctness of part K1189037.
Jinyang Liang did SDS page at teacher Yan Wang’s lab.



Jinyang Liang purified the ferritin.
Jinyang Liang and Qingdian Mu tested Berlin‘s parts again, there are only primer-dimers after PCR.
Jinyang Liang did PCR to get HL containing BamHI and HindIII sites. Renjie Shang digested HL fragment and pET-28a. Jinyang Liang purified the digest product and ran gel electrophoresis to estimated concentration of DNA. Then they did ligation and transformation.

Thermo-regulator

J23101 (J1) andJ23106 (J6) were got from 2015 kits, and were transformed into E. coli Top 10 strains which were spread on LB ager culture medium with relevant antibiotic later. The sequencing results match the correct one. Cun Wei extracted K1E, K2E, K3E, J1, J6 plasmids. Then ligations of J9 and K1E, K2E, K3E were down by Cun Wei after gel extraction and agarose gel electrophoresis Cun Wei did gel extraction after digestion andtransformed product of ligation into DH5α competent cells.

Fig.7. The target bands are around 1000bp. J9K1E, J9K1E 1075bp, J9K3E 1088bp. Sequencing result of J9K2E is not correct.

Fig.8. The target bands are around 1000bp, J9K2E 985bp.

Zhuo Pan succeed in making up Gibson assemblyMaster Mix and 1.33×Gibson assemblyMaster Mix. For details, please see the protocol.

InterLab

6.1
Four parts (J23108, J23109, J23111, I13504) was got from the 2015 Kit Plate 4, and transformed into E. coli K-12 DH5-alpha which were spread on LB ager culture medium with relevant antibiotic.
6.2
Spread out 1 plate per part. Incubate plates overnight at 37 degree centigrade and 220 rpm in 12mL tubes.
6.3
After plasmid extraction, these parts were double digested, J23108, J23109 and J23111 with SpeI (NEB) and PstI (NEB), I13504 with XbaI (NEB) and PstI (NEB).
After gel extraction, the promoter parts was ligated to I13504 with T4 DNA Assemble (Takara, Dalian) overnight.
6.4
These plasmids were transformed into E. coli K-12 DH5-alpha.
6.5
The colonies didn’t turn green under 450 nm light.
Trouble shouting but without result.
6.6
Traci toldusthe promoters they had us using (J23108, J23109, and J23111) all have two extra illegal SpeI sites in the plasmid which would make BioBrick assembly impossible.
Many thanks to Carnegie Mellon for finding this incredibly important problem.

Dry Lab Overview

(Theory Part) In order to find out whether our design of mag-receiver can work, Ruipeng Wei and Weiyan Chen simulated the temperature field around one single ftnA molecule. But unfortunately result showed no optimism. So we then searched deeply and got several new formulas to calculate temperature transmission.
(Device Part)For the sake of watching fluorescence better, Qikai Qin and Weiyan Chen decided to make a box named “Blue Light Generator” just for this usage. Qikai Qin ordered the blue LEDs whose wavelength is around 450 nm. The 450nm light is the second excitation wavelength of GFP, under which wavelength the GFP can stabilize for more minutes. At the meantime, Weiyan Chen and Ruipeng Wei made a solenoid.

• Week 8 (June 8 –June14)

Wet Lab Overview

We stopped testing Berlin’s parts this week and made the experimental procedure clear. There are some problems with the parts and we paused our InterLab experiments.

Mag-receiver

Jinyang Liang and Renjie Shang did PCR identification, the agarose gel electrophoresis shows that the plasmid is empty vector.
Jinyang Liang and Renjie Shang extracted plasmid, ran PCR to add enzyme sites to HL, purified enzyme product and did ligation and transformation. Theagarose gel electrophoresis shows that there are many bands on the gel including target band. The sequencing proved that the result didn’t match the correct sequence.
We had not got the correct and sequenced part when we ran out of plasmids.
Qingdian Mu and JieLian started the protein expression experiment to study the expression of pET-ftnA. We used ammonium ferric citrate as iron source with iron final concentration 0.5mM.

Thermo-regulator

Cun Wei extracted K1E, K2E, K3E, J1, J6, plasmids. Then He digested K1E, K2E, K3E with XbaI and PstI and digested J1, J6 with EcoRI and SpeI. Ligations of J1 and K1E, K2E, K3E;J6 and K1E, K2E, K3E were down by Cun Wei after gel extraction and agarose gel electrophoresis Cun Wei did gel extraction after digestion andtransformed product of ligation into DH5α competent cells .

Fig.9. The left lane is DL2000 marker, J1K1E, J1K2E, J1K3E, J6K1E, K6K2E, K6K3E are expected at 1000bp.

Zhuo Pan extracted plasmids loaded parts I0500, I13504, J04450. I0500 was digested with EcoRI and SpeI, I13504 was digested with XbaI and PstI, J04450 was digested with EcoRI and PstI. Ligation was down without gel extraction. He use red white spot screening to screen the target strains. However, there are no colonies on the plate.

Dry Lab Overview

(Theory Part) Ruipeng Wei calculated the temperature field around one single ftnA molecule for the second time and got a good distribution about the temperature.She then used this method to calculate the whole cell temperature distribution with every ftnA molecule fixed in the cell environment.
(Device Part)This week we got two opposite news, one is that we went well on the fluorescence-watching box; while the bad news is that we finally found it impossible for us to build an amplifier with such a high power.

• Week 9 (June 15 –June21)

Wet Lab Overview

This week we designed primers that would be used to construct T7 platform. The total project for experiments was formed.

Dry Lab Overview

(Theory Part) Because protein molecules are randomly distributed in the cell and move around stochastically, Weiyan Chen and Ruipeng Wei added a parameter normally distributed in (0,1) onto the equations to describe molecules’ movement in every some time interval and calculated the temperature field of the whole cell again. But after debugging for several times we found it did not show a good result yet.
(Device Part)Weiyan Chen investigated several companies who produce the signal generator and amplifier, and visited many senior advisorsand professors to solve the problem about high power.
In the meanwhile, Qikai Qin and Weiyan Chen successfully made the device of observing the GFP or RFP. The Blue Light Generator was born this week.

• Week 10 (June 22 –June28)

Wet Lab Overview

We stopped all experiments to prepare the final exam.

Dry Lab Overview

(Theory Part) This week, wet lab members have found ferritin’s unexpected leakage. We consider it to be the lacI binding sites on the PQE vector that caused the problem. So Ruipeng Wei used a novel method based on partition function to explain the leakage.
(Device Part)After so many investigations and analysis, we at last gave up the work of making an amplifier.

• Week 11 (June 29–July 5)

Final examination was from July 3 to July 12.

• Week 12 (July6– July12)

Wet Lab Overview

Final examination was from July 3 to July 12. Cun Wei started experiment after finishing the exam.

Mag-receiver

Cun Wei did three PCR to get light chain, heavy chain and GSlinker. Cun Wei overlap the GSlinker and heavy chain, then Gibson assembly of light chain and linker-H was down.
Cun Wei did three consecutive PCR to get HL fragment with prefix and suffix.
Cun Wei get ftnA fragment containing prefix and suffix by two PCR.

• Week 13 (July13– July19)

Wet Lab Overview

We started experiments following total project. Qikai Qin and Jinyang Liang restarted the InterLab Study. The three new devices were under construction.

Mag-receiver

Number 1 stands for construction of pSB-L-GSlinker-H
Number 2 stands for construction of pSB-HL
Number 3 stands for construction of pSB-ftnA
Number 4 stands for construction of pQE-L-linker-H
Number 5 stands for construction of pQE-HL
Number 6 stands for construction of pQE-ftnA
1.
Qingdian Mu extracted pQE-80L, pUC57-GSlinker, pUC57-RiboJ and K1189037, J04450. Qingdian Mu amplified the L-linker-H by PCR, added RBS to L-linker-H by PCR, added prefix and suffix to L-linker-H by PCR.
Cun Wei did three consecutive PCR to get light chain, heavy chain and GSlinker. Cun Wei overlap the GSlinker and heavy chain by PCR, then Gibson assembly of light chain and linker-H was down.
2.
Cun Wei digested HL generated last week with XbaI and PstI. Cun Wei did three consecutive PCR to get HL fragment with prefix and suffix.
3.
Cun Wei digested ftnA generated last week with XbaI and PstI. Qingdian Mu digested pSB-R0011 with EcoRI and SpeI, assembled J04450, R0011 and ftnA
Cun Wei get ftnA fragment containing prefix and suffix by two consecutive PCR.
4.
Same pre-product with Numb 1 Part.
5.
Cun Wei did PCR to get heavy-light chain fragment (HL), and then he did double enzyme digest of PCR product and pQE80L, ligation and transformation. There are no target bands. Maybe it is due to the lack of gel extract.
6.
Cun Wei digested pET-ftnA and pQE80L, then ligation and transformation were down. There are no target bands. Maybe it is due to the lack of gel extract.
Jie Lian and Renjie Shang practiced SDS-PAGE and Ni-sepharose purification and summed up the experience.

Thermo-regulator

To insert VMA into 601 site of T7 RNAP, Zhuo Pan transformed the ordered plasmid pUC-VMA into Top 10 cells, then he extracted the plasmid for PCR. Two PCR were down to get VMA fragment and VMADEAD fragment. Zhuo Pan extracted plasmid that loaded T7 RNAP gene, splited T7 RNAP gene by PCR at 601 site. Gibson was down to assemble N-terminal of T7 RNAP gene, C-terminal and VMA or VMADEAD. We did PCR identification of the product of Gibson, the Agarose gel electrophoresis shows bands at 2.7 K which stands for intact T7 RNAP gene. The experiment was failed.
Xihan Zhang extracted I0500, I13504, and J04450. Jinyang Liang digested I0500 with EcoRI and SpeI, I13504 with XbaI and PstI, J04450 with EcoRI and PstI. Ligation was down after gel extraction. Xihan Zhang transformed the products into DH10B competent cells. Xihan Zhang did PCR identification. The electrophoresis shows that there are no bands when using primers VF2 and VR. However, there are target bands when using the primers designed by ourselves. It’s indicate that there are multi-sites binding exits.
Xihan Zhang and Jinyang Liang repeated the ligation and transformation. They use two pairs of primers to identify the ligation, the first pair is seg2-F and VR, the second pair is VF2 and seg2-R.
The electrophoresis shows there are target bands above 2000bp. The corresponding bacterium was inoculated to LB liquid medium containing arabinose and it fluoresced !

Fig.10. The sequencing results match the theoretical sequence.

Dry Lab Overview

(Theory Part) From several references Ruipeng Wei concluded a theoretical formula fit for the temperature distribution calculation with stochastic movement. We did another more simulationand finally got a good result. From that we could conclude that ftnA is possible to heat the whole cell. Ruipeng Wei also did the sensitivity analysis of the ftnA’s deterministic model to find out every parameter’s importance of the model.
(Device Part)For a better view of bacteria attraction experiment, Xihan zhang designed a device made of acrylic plate whose core is a six well plate struck by six magnets. She called it “Captor beta”.

Captor beta

• Week 14 (July 20– July26)

Wet Lab Overview

The ordered gene synthesis productions arrived. They are seg 1 to seg 5, which were used for constructing T7 platform.

Mag-receiver

Qingdian Mu did PCR identification of pQE-L*H, pQE-HL, pSB-ftnA and pSB-HL, no target bands. The sequencing of pQE-ftnA shows that the result sequence doesn‘t match the correct one.
Circuits 1 to 6 was under construction by Cun Wei and Qingdian Mu, the main problems were that more than one bands appeared after amplificating Gibson product, ligation was failed even with gel extraction.
Renjie Shang succeed in ferritin purification, proved by SDS-PAGE. To achieve an optimal induction concentration for Pet28a harboring ftnA, Jie Lian designed more IPTG concentration gradient from 0.1mM to 1mM. Considering the expression quantity and toxic damage to cell, we decided the final induction concentration as 0.8mM.
Jie Lian examined the supernate and purification product with Prussian blue staining and native-page. There was no color change for the concentration of samples was low.

Thermo-regulator

Zhuo Pan did PCR to get seg 1 to seg 5 fragments. Xihan Zhang did the agarose gel electrophoresis of five fragments and digested pSB1C3 to estimate the concentrations. Five fragment and the backbone were assembled by Gibson assembly. Xihan Zhang did the transformation and the bacterium were spread out on the plate with arabinose. The colony turned green on the plate. However, there no target bands on the gel after PCR identification. Besides, we transformed the plasmid into DH10B, the strain doesn’t turn green anymore. We failed to construct the T7 platform. We thought that there was incomplete plasmid containing GFP in E.coli, that’s why the strains turned green.

InterLab

Xihan Zhang, Qikai Qin decided to add Ribo J into the circuits and ordered it from GENEWIZ.
7.20
Three parts (J23101, J23106, J23117) was got from the 2015 Kit Plate 4, and transformed into E. coli K-12 DH5-alpha which were spread on LB ager culture medium with relevant antibiotic.
7.21
Spread out 1 plate per part. Incubate plates overnight at 37 degree centigrade and 220 rpm in 12mL tubes.
7.22
After plasmid extraction, these parts were double digested, J23101, J23106 and J23117 with SpeI (NEB) and PstI (NEB).
After PCR, linear I13504 with suffix and prefix was got, then was double digested with XbaI (NEB) and PstI (NEB).
The promoter parts was ligated to I13504 with T4 DNA Assemble (Takara, Dalian) overnight.
7.23
These plasmids were transformed into E. coli K-12 DH5-alpha.
7.24
The colonies didn’t turn green under 450 nm light.
Trouble shouting.
7.25
Qikai Qin and Jinyang Liang decided to obtain I13504 through plasmid extraction.
Growing the E. coli K-12 DH5-alpha containing I13504 (37 degree centigrade, 220rpm).
7.26
Jinyang Liang did the plasmid extraction of I13504.
Qikai Qin did the double digestion with XbaI (NEB) and PstI (NEB).
Jinyang Liang did the ligation of I13504 and these promoter parts.

Dry Lab Overview

(Theory Part)This week we got some statistics about GFP expression with pBAD. So Weiyan Chen and Ruipeng Wei did the statistic analysis and compared it with our theoretical model. And they fitted well.
Weiyan Chen also constructed the stochastic model of ftnA based on the Gillespie Algorithm. Then Ruipeng Wei adjusted ftnA’s molecules in temperature distribution model with the newest calculated results.
(Device Part)Xihan Zhang considered to add the Blue Light Generator into “Captor beta” and redesigned the device.

Captor Design

• Week 15 (July 27– August2)

Wet Lab Overview

This week we debug the experimental design and make a clearer division.

Mag-receiver

This week we debug the experimental design. The improved design as follows .

Construction of pQE-L*H

Three PCR are carried out simultaneously
1.
Template: K1189037
Aim: get light chain fragment
Primers: L-F BamHI and B-R
2.
Template: pUC57-GSlinker
Aim: get GSlinker fragment
Primers: C-F and C-R
3.
Template: K1189037
Aim: get heavy chain fragment
Primers: D-F and D-R

Two PCR are carried out simultaneously
1.
Template: light chain fragment
Aim: adding lap region
Primers: L-F lap and B-R
2.
Template: heavy chain fragment
Aim: adding lap region
Primers: D-F lap and CD-R lap

Agarose gel electrophoresis
Purification of PCR product or Gel extract
Double enzyme digest of pQE-80L
Gibsom assembly
Transformation

Construction of pSB-L*H

Three PCR are carried out simultaneously
1.
Template：k1189037
Aim: get light chain fragment
Primers: L-F BamHI and B-R
2.
Template：pUC57-Gslinker
Aim: get Gslinker fragment
Primers: C-F and C-R
3.
template：k1189037
Aim: get heavy chain fragment
Primers: D-F and D-R

Agarose gel electrophoresis
Purification of PCR product or Gel extract
Agarose gel electrophoresis
Gibson assembly
PCR：adding prefix and suffix to L-linker-H
Agarose gel electrophoresis
Gel extraction
Double enzyme digest of PCR product
Purification of enzyme digest product
Agarose gel electrophoresis
ligation
Transformation

PCR：adding RBS to L-linker-H
Agarose gel electrophoresis
Purification of PCR product or Gel extract
PCR：adding prefix and suffix to RBS-L-linker-H
Agarose gel electrophoresis
Gel extraction
Double enzyme digest of PCR product
Purification of enzyme digest product
Agarose gel electrophoresis
ligation
Transformation

Construction of pQE-HL

PCR:
Template: k1189037
Aim: get heavy-light chain fragment(HL) that containing BamHI and HindIII sites
Primers: 037pQE-Fand 037pQE-R

Agarose gel electrophoresis
Purification of PCR product or Gel extraction
Double enzyme digest of PCR product and pQE80L
Agarose gel electrophoresis
Purification of PCR product or Gel extraction
Agarose gel electrophoresis
ligasion
Transformation

Construction of pSB-HL

PCR1:
Template: k1189037
Aim: get heavy-light chain fragment(HL) that containing suffix
Primers: 037pSB-1Fand 037pQE-1R
PCR2:
Template: product of PCR1
Aim: adding RBS to HL
Primers: 037pSB-2Fand 037pQE-2R
PCR3:
Template: product of PCR2
Aim: adding prefix to RBS-HL
Primers: 037pSB-3Fand 037pQE-3R
Gel extraction
Double enzyme digest of PCR product and plasmid J04450
Purification of enzyme digest product
Agarose gel electrophoresis
ligasion
Transformation

Construction of pQE-ftnA

Extraction of pET-ftnA and pQE80L
Double enzyme digest with BamHI and HindIII
Agarose gel electrophoresis
Gel extraction
Agarose gel electrophoresis
ligasion
Transformation (DH10B competent cells)

Construction of pSB-ftnA

PCR1: add RBS when getting ftnA by PCR
PCR2: add prefix and suffix to RBS-ftnA
Double enzyme digest of PCR product and J04450
Gel extraction
Agarose gel electrophoresis
ligasion
Transformation (JM109 competent cells)

Jie Lian went on the SDS-PAGE, trying to identify an optimal IPTG concentration for pQE80l induction, but always failed for there was no apparent targeted band at 20~21kDa. For characterizing the magnetization effect, Renjie Shang added 3 milliliters of cell suspension (BL21 harboring pet28a-ftna and DH5α harboring pqe80l-ftna, bacteria were cultured in LB medium inoculated with 1mM ammonium ferric citrate to dishes above a Rubidium iron boron magnet.



The result of pet28a group was apparent while pqe80l group could see no difference. After analyzing, we speculated that ftnA can’t correctly express in the wrong host cell DH5α.
We tried more iron containing medium to incubate BL21 harboring pet28a-ftna and found that ferrous sulfate can result in more apparent pattern than ammonium ferric citrate.

Thermo-regulator

Although we had been constructing the plasmid more than one week, we didn’t get positive results. We thought that the PCR would trigger mispairing and that’s why we fail to assembled the fragments. So we started doing gel extraction to purify the products of PCR and concentrated the products with concentrator. We repeated the chart for seven times, but we still didn’t get positive results.

InterLab

7.27
The constructed plasmids was transformed into E. coli K-12 DH5-alpha by Jinyang Liang.
7.28
Weiyan Chen and Qikai Qin used the blue light generator to observe the expression of GFP, and found that the fluorescence of J23101 and J23106 was obviously stronger than J23117.
Then the three devices were preserved.

Dry Lab Overview

(Theory Part) Weiyan Chen constructed the stochastic model of T7 platform based on the Gillespie Algorithm. Ruipeng Wei adjust several parameters in the two stochastic models.
(Device Part)Xihan Zhang completed the new design of “Captor” and connected a company to accomplish it.

• Week 16 (August3– August9)

Wet Lab Overview

We tried to construct T7 RNAP plasmid in stages.

Mag-receiver

Cun Wei successfully constructed the pQE-ftnA in DH5α.
Qingdian Mu did three parallel experiments for constructing pQE-HL, only the first batch of bacterium grow on the plate. However, there are no bands on the gel after PCR identification. We found that the rTaq enzyme was inefficiency later.
Cun Wei was constructing pQE-L*H, there are only primer-dimers after PCR identification.
Cun Wei transformed Pqe80l-ftn from DH5αinto DH10B. After getting the sequencing data, the induction experiment was succeed with final concentration 1mM for Pqe80l.
The protocol of ni-sepharose purification was optimized. But Native-page and prussian blue staining still didn’t work.

Thermo-regulator

We thought it’s hard to assembled 6 fragments for Gibson assembly. So we tried to do it in three steps, that is assembling seg 1, seg 2, seg 3 first, then the seg 4 and seg 5, finally the plasmid would be assembled.
We didn’t get positive results, although the PCR identification before transformation is positive.

InterLab

8.2
Qikai Qinstartedtoadd Ribo J intothethree InterLab devices.
Jinyang Liang transformed BBa_I0270 and BBa_R0040 into E. coli K-12 DH5-alpha.
8.3
Qikai Qin transformed the three devices with Ribo J into E. coli K-12 DH5-alpha. The first step is ligation of Ribo J and BBa_I13504, then the promoter was ligated in the front.
8.5
The sequencing results showed that only one device (BBa_J23117 + Ribo J + BBa_I13504) was correct.

Dry Lab Overview

(Theory Part) With the ftnA’s stochastic model established, Weiyan Chen calculated its intrinsic noise from which we found ftnA’s expression amount was stable. It means that the heating effect can be really stable in a long time interval and supported our temperature distribution model well.
(Device Part) “Captor” was under producing!

• Week 17 (August10–August16)

Wet Lab Overview

Xihan Zhang, Qikai Qin, Weiyan Chen and Renjie Shang participated in summit hosted by PKU iGEM in Beijing.
We visited Changqian Cao in Beijing, and we are told that it’s better to choose ftnA because the chassis is E.coli. We additionally discussed the project schedule and finally decided to abandon the other circuits.
We decided to have a collaboration with NYU_Shanghai.

Mag-receiver

Qingdian Mu did parallel experiments as follows.



We changed the project before PCR identifications were completed down.
Pqe80l was proved worked well. This week Jie Lian and Renjie Shang incubated bacteria with 7.125mM ferrous sulfate. However the unexpected positive results were observed in most of the control groups. We started to suspect the mag-attraction test. Jie Lian concentrated the ferritin purification product with Amicon Ultra-15ml (Millipore) and went on with Native-page and Prussian blue staining. No positive result.

Thermo-regulator

We repeated the experiments of constructing T7 platform.
We decided to have a collaboration with NYU_Shanghai. Qikai Qin measured the GFP expression under pBAD promoter with different concentration of arabinose. Finally, we gained the optimal induced concentration of arabinose on LB solid medium.

InterLab

8.11
Qikai Qin grew the three devices, positive and negative control, the all of them are in three biological replicates. And he prepared the extra device (BBa_J23117 + RiboJ+ BBa_I13504), and M9 media and different concentration of sodium fluorescein.
8.12
Qikai Qin use the plate reader to test the expression of GFP under Jing Wang’s help in absolute unit. In the night, he processed data.
8.13
Qikai Qin discussed the protocol of Raman Spectrometer with Yetian Su.

Dry Lab Overview

(Theory Part) Using the result of T7 platform’s stochastic model, Weiyan Chen and Ruipeng Wei calculated its intrinsic noise from which we can concluded that the reporter protein(GFP) expression level had a stable steady-state however it was not so sensitive to the splicing efficiency or the amount of T7 polymerase because the high activation of T7 polymerase.
(Device Part) “Captor” was under delivering! Qikai Qin ordered some new LEDs with the wavelength of 450nm and 580nm.

• Week 18 (August17– August23)

Wet Lab Overview

The T7 RNAP experiments failed.

Mag-receiver

Renjie Shang and Qingdian Mu went to Changqian Cao’s laboratory in Beijing to do ferritin experiments. We did the experiments following the ferritin protocol except that we induced gene expressing at 30℃ and we used ferrous sulfate with final concentration 7.125mM.

Fig.11. The SDS results is not satisfactory for supernatant is vague on the gel. The native page was failed that there are not blue band on the gel .

Transmission Electron Microscopy (TEM) Test and Magnetization Curves are obtained in Beijing.
Attempting to figure out the ferritin concentration per cell, Jie Lian went to Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) to search for Yetian Su's guidance. The absorption curve can show the obvious difference of ferritin expression between induced cell and control but failed in quantifying the concentration we want.

Thermo-regulator

We used commercial Gibson solution to assemble the fragments, the results were negative. We abandoned the T7 RNAP experiments.

InterLab

8.20
Qikai Qin and Weiyan Chen went to Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT), Chinese Academy of Sciences (CAS). They prepared the samples and ready to use Raman Spectrometer.
8.21
Under Yetian Su’s guidance, we completed the test with Raman Spectrometer. But the data was not ideal. Then we had a discussion about it.

Dry Lab Overview

(Theory Part) In the CCiC, we communicated with HFUT-software iGEMers and knew that they needed some assistance in the simulation part of their software. So we helped them to improve their functions of the simulation part and deal with some detailed statistics. Weiyan Chen also concluded the common use of ODE equations and logistic model and assisted them of the parameter choice and analysis.
(Device Part)We got our device and tested it with the bacteria attraction experiment. Qikai Qin and Weiyan Chen added the new LEDs in the device and took some specimen pictures.

Captor

• Week 19 (August24 – August30)

Wet Lab Overview

This week we designed a big experiment for Mag-receiver subteam. We prepared 32 Erlenmeyer flasks for the experiment, when we were going to inducing gene expression, the problem arised: the temperature in big incubator couldn’t down to 18℃. We have to abandon the original plan.

Mag-receiver

Renjie Shang repeated the experiment from cultivation in Beijing, but using ammonium ferric citrate with final contrition 1mM, the SDS results were still poor and native page fail again.
Qingdian Mu returned to Qingdao, summarized the protocol of ferritin experiment, got down to doing the experiment following the new protocol. The electrophoresis device for SDS page in our lab is hard to operate, so we asked teacher Yan He for help. We were permitted to use electrophoresis device at her lab.
This week we designed a big experiment for Mag-receiver sub team. We prepared 32 Erlenmeyer flasks for the experiment , when we were going to inducing gene expression, the problem raised: the big incubator couldn’t down to 18℃. We have to abandon the original plan.



This week the main problem was that native page didn’t develop color. We speculated that the protein concentration was not enough.
The first preliminary experience prepared for ferritin concentration per cell quantification was conducted. At first Jie Lian measured the ferritin purified product prepared for adjusting the optical density quantitation protocol with BCA protein quantitation kit. But the data was not ideal because the measurement error was big.

Thermo-regulator

Zhuo Pan transformed J1K1E, J1K2E, J1K3E, J6K1E, J6K2E, and J6K3E from pSB1A2 into pSB1C3. He digested J1K1E, J1K2E, J1K3E, J6K1E, J6K2E, J6K3E and J04450 with EcoRI and PstI. Ligations were down after gel extraction. The sequencing results proved the experiment was successful.

InterLab

8.26
Qikai Qin used Flow Cytometer to test the expression of GFP under Yu Ma’s help. They processed data together.
8.27
Qikai Qin submit the InterLab worksheet and InterLab Protocol.

Dry Lab Overview

(Theory Part)This week, we got ftnA’s magnetic properties test result and changed related parameters in the heating power of ftnA and temperature distribution model and updated our simulation results.
(Device Part)Qikai Qin and Xihan Zhang optimized the light intensity of “Captor” and got better views.

• Week 20 (August31 – September6)

Wet Lab Overview

We validated the iron core in the ferritin.

Mag-receiver

This week, Native page was successful using concentrated ferritin which from 900mL bacterium suspension .



Jie Lian prepared the ferritin purified product with final concentration about 1.2mg/ml and mineralized samples. Under Jian’an Wu’s assistance, Jie Lian and Weiyan Chen test the temperature change in samples under alternating magnetic field with fiber temperature detector in Laboratory of Innovative Medical Technology of Tsinghua University. Unfortunately there was no obvious temperature change. Dr. Wang explained that it is hard to observe the heated effect of ferritin solution.

Thermo-regulator

The experiments about thermal noise was in process.

Dry Lab Overview

(Theory Part)Ruipeng Wei and Weiyan Chen checked every parameter in every model and found either deductive reasoningor sufficient evidence for them.

• Week 21 (September 7 – September 13)

Wet Lab Overview

Most experiments were stopped.

Mag-receiver

Jie Lian quantified ferritin concentration per cell with electrophoresis image analysis system.

Dry Lab Overview

(Theory Part)Preparing for the wiki page.
(Device Part) Preparing for the wiki page.

• Week 22 (September 14 – September 18)

Finished the wiki. We’re looking forward to the meeting in Boston.

Cistrons Concerto

Thanks

1.Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences

2.NEW ENGLAND Biolabs

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E-mail: oucigem@163.com

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