Team:XMU-China/wetlabjournal

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Many team members were away for internship in another city. Experiments progress became very slow.

Week 14: July 9th to July 15th

Monday, July 9th
This week, another three of the team members were away for internship in Zhangzhou Campus. It meant only three members were available for the experiment.
Colonies were grown well in the morning. Pick one colony of each plate and cultivated.
After 12 hours we isolated the plasmids.

Tuesday, July 10th
Plasmids from the previous day were digested and ran on a gel for restriction analysis, however the result of the gel indicated the ligation was not totally successful: RBS-gfp(LVA)-TT should be reconstructed. So we pick another colony from the plate.

Wednesday, July 11th
Prepared the competence BL21 cells and transferred the succeed ligation product: PBAD-RBS-cI(LVA)-TT, RBS-tetR (LVA)-TT. Isolated the plasmids of RBS-GFP (LVA)-TT and digested for restriction analysis. After running on a gel, the plasmid turned to be right.

Thursday, 12th
Pick one colony

all of the previously transformed and isolated parts were also used to run an agarose gel confirming the identity of the transformed parts.

Week 15: July 15th to July 22nd

Monday, July 16th
 Wiki:
SUNNY MONDAY!! Got up at 8:00AM and started a new week in lab. Wang realized the fact that it would be a huge project to translate all the experimental journals into English in last few weeks. So she started writing the new journals in English and translating the original ones.
 Experiments:
Goal: Pcons+RBS-tetR-TT, P_BAD+RBS-cI-TT
At 10:00AM, no growth/colonies were observed on the plates. However, by the late afternoon, colonies were visible on all of the plates, so picked one colony and cultivated it.
Plasmids isolated and digested for ligation. BSA was added in XbaI system as description. After 1 h of digest, we  ran on a gel. An unanticipated band revealed the possible mistake of target gene: the band of plasmid with BSA addition was wrong. We planed to test the BSA the next day and do another digest for restriction analysis. To speed up, we would perform ligation if the restriction analysis was correct.
 Meeting:
Team meeting at 7:00PM, Leader Wu divided all the members into different groups and announced the assignments for each group: Experiment, Wiki, Human Practice, Management, Communication. He also reiterated the experimental disciplines. Wang summarized the experiments and pointed out our next steps: finish all the construction in two weeks and characterize our device.

Tuesday, 17th
Experiments:
 Isolated the plasmids and digested for analysis and ligation as well. We ran on a control test for BSA. The result of gel indicated that problems existed both on BSA and the bacteria we preserved, which seemed had been contaminated.

Wednesday, 18th
Experiments
Goal: To verify all the bacterial glycerol stocks and the transformation.
Experiment:
Isolated the plasmids, digested for restriction analysis and ran on a gel. The result was frustrating: they were all wrong! We should perform ligation again as soon as possible.

Thursday, July 19th
Experiments:
Goal: To ligate PcI+Rgfp(LVA)TT, Pcons+RtetR(LVA)TT
Isolated the plasmid and digest for ligation. Ran on a gel and performed gel extraction. After 4h of ligation, transferred into competence BL21 which were prepared in July 16th.
Made plates with Amp

Friday, July 20th
Colonies were still invisible until late afternoon. Exciting news was that the colonies of PcI-RBS-GFP(LVA)-TT had    fluorescence, which indicated the possible successful of transformation. But the other one was not good: colonies could be observed only in one plate. In the evening, picked colonies and cultivated them. 
  
Saturday, July 21st
Experiments:
Goal: To verify if the transformation was successful.
Plasmids isolated and digested for restrict analysis. The result was upset but we still stuck at it - after all, the bacterial liquid culture was illuminating. So picked another two colonies in the plate and hoped for the best.

Sunday, July 22nd
Experiments:
Goal: To verify again whether the transformation was successful and prepared to performing another ligation.
The bacterial liquid had fluorescence, both of them! This exciting news had raised our hope. Isolated the plasmids and digested for restrict analysis. The result indicated that one of the bacterial culture was correct! So added 100 μL aliquot into LB, attempting to make bacterial glycerol stocks. However, strange thing was that liquid culture had not illuminated! Added another 100μL aliquot into LB and put two LB in the shaker at 37°C.

Week 16: July 23rd to July 29th
Monday, July 23rd
Experiments:
Isolated the plasmid of original correct bacterial in 21th and two liquid culture in 22nd from the original one. Ran on a gel for restriction analysis. The result was beyond belief: only the band of  original one was correct, but it seemed that once we add 100μL aliquot of original bacteria into new LB, the band went wrong. We cannot figure out why this strange thing could happen.
In the evening after the team meeting, streaked plate with original 7.21 liquid culture.

Team Meeting:
Wang summarized the experiments in the last week. It was a tough task building the same two gene circuits again and again. Huang and Yu presented her communication with 2012iGEM members in Peking University and University of Science and Technology of China. She pointed out some shining part of them, which we could learn from and improve our own team. Mo shared his idea on an article he read and discussed the method for determining the fluorescent intensity. Chi showed the progress in Human Practice: we had prepared abundant materials and waiting for Xie's return. Fan and Yan proposed their design in wiki and mascot, and everyone were so exciting when discussing how mascot should be like.

Tuesday, July 24th
Experiments:
Isolated the plasmid and ran on a gel for restriction analysis.
Made PBS for determining the fluorescence.

Wednesday, July 25th
Experiments:
Added 1mL aliquot into 200mL new LB and put in the shaker at 37°C. After 2 hours of shaking, added 2.7mL arabinose to each liquid culture. Determined the fluorescence value and OD600 every hour. We all learned how to use the equipment. The values of unstable GFP were too low, which made us very nervous: those circuits we spent so much time to made might become nonsense in a second! We quickly isolated the plasmid for restriction analysis and ran on a gel. The result indicated that the bacterial glycerol stocks may be contaminated, which turned out to be the best bad news for us.

Thursday, July 26th
Experiments:
Goal: To do another ligation of constructive promoter and RBS-tetR-TT once again, to verify the streak plate.
Isolated plasmids and ran on a gel. The result for ligation was perfect: the band was neat and clear. However, that for analysis was not really good. After gel extraction, did gel ligation and put it in 4°C, waiting for transformation.
Design:
Mo, Qiu and Yan managed to sewed a sample of our mascot. They brought needles, thread, cloth magnet and cotton to lab. Qiu even wore a rubber glove, claiming that a stronger friction force could be helpful in sewing, or maybe she was just used to wear it when performing experiments.

 

Friday, July 27th
Experiments:
Goal: To verify if all the glycerol stocks of PBAD-RBS-gfp(LVA)-TT are still correct.
Since we had found the high risk of mutation for liquid culture due to the transfer of culture, another method for preserving was badly needed. Also we should verify if the glycerol stocks are still remain the same.
Isolated the plasmid for restriction analysis then ran on a gel. The result indicated that three of four stocks had the possibility being wrong. Streaked a plate from the right one.

Design:
Mascot had almost finished, only problem here was that it cannot sit or stand by itself. Maybe thicker legs and heavier body can solve this problem.

Saturday, July 28th
Experiments:
In order to verify part PcI-RBS-gfp(LVA)-TT and ligate this part to others, did another digest for restriction analysis and ligation. Sadly, the gel result was still disappointing: three bands were observed after double digestion. But two of the bands revealed that they might be correct, yet being contaminated. So Advisor Wu decided to extract gel with correct bands and perform ligation.

 

Sunday, July 29th
Experiments:
Still stuck at PcI!!! Isolated plasmid from the streaking plate from original transformation one. Digested for restriction analysis and ran on a gel. The result was still not pleasant. Taking advice from another graduate student Zheng, diluted the liquid culture in 10-6 and 10-7 times and spread on LB Agar with Amp.

August
Week 17: July 30th to August 5th
Monday, July 30th
Experiments:
Digital Display:
Goal: ligate Pcons-RBS-tetR-TT + PtetR
In the morning, we started on the first try. After plasmid isolation and digestion, ran on a gel and result was still sad. So we try again in the late afternoon and performed ligation.
The colonies in the plate of the previous day grew well, so picked one colony of each plate and streaked lines at the same time.

Team Meeting:
Wang summarized the genetic circuit parts finished last week, and restated the essentials of attitude and carefulness when conducting experiments. Xie presented his communication with iGEMers in The Hong Kong University of Science & Technology. He communicated with the team members and have a pleasant discussion.

Tuesday, July 31st
Experiments:
Digital Display:
Goal: To verify all the plate streaked or spread in July 29th.
Isolated plasmids and ran on a gel. The result was not perfect but still satisfying. One of the ligation turned out correct.
Sophia sent everyone an e-mail, pointing out the time limitation and rest tasks of experiments. The lab team was divided into three groups in charge of digital monitor, time delay and cell immobilization, respectively.

Wednesday, August 1st
Experiments:
Goal: To ligate RBS-tetR-TT with PcI-RBS-gfp(LVA)-TT
Isolated plasmids and digested for ligation. After running a gel,  bands appeared not having been digested completely. Still did the ligation anyway.

Cell Immobilization:
We started thinking about methods to establish our numeric display device and to bring our engineering bacteria into operation. Obviously, the foremost thing we should do is to fix the engineering bacteria from freely suspended cultivation to the tubes of our device, namely cell immobilization. Meanwhile, cultivation condition and fluorescence intensity of immobilized cells should be made certain.
After studying literatures and asking senior students for help, we decided to explore three kinds of methods of immobilizing our engineering bacteria: 1) calcium alginate beads; 2) PDMDAAC-NaCS microcapsules, 3)  intra-hollow Ca-alginate capsules.

 

Thursday, August 2nd
Experiments:
Goal: To verify Part I and perform ligation if Part I is right.
Isolated plasmids and digested both for restriction analysis and ligation. The result was so upsetting: the construction of Part I was still unsuccessful. Better try another ligation next day.

Cell Immobilization:
Added 100uL PBAD-RGT aliquot into LB medium with Ampicillin，37°C shaker incubated for 12h.
Goal: To test if PDMDAAC will restrain growth of the engineering bacteria.
As the graph shows, In the first 90 min, OD was relatively high ( in a normal value), but then it declined dramatically. 

Friday, August 3rd
Experiments:
Digital Display:
Goal: To build Part I
Plasmids isolation, digestion and gel extraction all went smoothly. Part II-III-IV was correct.

Cell Immobilization:
Goal: To prepare calcium alginate and embed engineering bacteria with plasmid PBAD-RGT.
Prepared: 3% sodium alginate solution; 0.05 mol/L CaCl2 solution; sterile water.
Centrifuged the cultivated sample for 1min at 6000rpm to collect the deposit (about 2~3g). Resuspend the cells with sterile water and centrifuge for 1min at 6000rpm，and drained off the supernatant. Mixed the cells with sterile water in a mass ratio of 1:2, then added equivalent volume of sodium alginate solution, mixing thoroughly.
Drew the  mixture in a 10mL injector, then dropped it one by one through the pinhead into 100mL  CaCl2 solution,  stirring at the same time. Laid it aside for 2 hours. Then drained off  the CaCl2 solution and rinsed the calcium alginate beads with sterile water for 2 or 3 times. Added 100mL  CaCl2 solution, soaking the beads overnight.

 

Saturday, August 4th
Digital Monitor:
Made competence BL21 and transformed ligation product of the previous day.

Cell Immobilization:
Goal: To prepare Sodiumcellu-losesulfate(NaCS) for making PDMDAAC-NaCS microcapsules.
Today we started learning to prepare NaCS, with the help of a senior.
Prepared: sulfuric acid and ethanol solution(1.51:1) maintained at 0℃; dry absorbent co/p> tton maintained at 60℃; industrial alcohol; deionized water; NaOH solution.
Immersed 4g  dry absorbent cotton in the solution in ice-bath for 66 min. Then squeezed out the solution and rinsed the reacted linters with alcohol in draught cupboard. Put it in 400mL deionized water and regulated pH to about 3. Stirred and dissolved it for 10 min then filtrated it. Collected the filtrate and regulated pH to 9.3 accurately. Added industrial alcohol, 1~1.5 times of the filtrate, until there appeared white retiary floccule. Centrifuged the floccule for 5 min at 5,000rpm and collected it. 65°C drying for more than 24 h.

Time delay:
Goal: To do the ligation of RBS-GFP-TT with the biobricks RBS1.0 and GFP-TT which we already have.
Isolate plasmids and ran on a gel. The result for analysis was not good for the bands of RBS were not clear. The result for ligation was not good for there are four bands of GFP-TT, two of them are correct. So we extracted gel and did gel ligation and put it in 4°C, waiting for transformation.

 Sunday, August 5th
Digital Display:
Goal: ligate Part III-IV and Part V-IV

Cell Immobilization:
PBAD-RGT beads emitted fluorescence.
Prepared calcium alginate and embed engineering bacteria with plasmid PcI-RLT.
PcI-RLT beads emitted fluorescence in CaCl2 solution.

Time delay:
Pick single colonies of luxI, luxR, luxpR.

Week 18: August 6th to August 13th

Monday, August 6th
Experiments:
Digital Display:
Goal: To verify if the transformation is successful and perform ligation once it does.
After cultivated in liquid LB for 12 hours, bacteria were collected in 1.5mL sterile tube and isolated plasmids. Digested for both restriction analysis and ligation then ran on a gel. The image showed that transformation was almost successful, still two of them appeared to be contaminated. We hope those extra bands were due to some careless mistakes during isolation process, not transformation.

Cell Immobilization:
Transferred the PcI-RLT  beads into  LB medium with Ampicillin, 37°C shaker incubated.
PcI-RLT beads emitted fluorescence, which revealed more obvious under ultraviolet light. The fluorescence didn’t trail off after cultivated for several hours.
Goal: To prepare PDMDAAC-NaCS microcapsules.
Got 2.17 g NaCS.
Before making the microcapsules, we prepared 6% PDMDAAC solution and NaCS solution (the concentration was not sure ). For ensuring the concentration of NaCS solution we need, firstly we dissolved 0.4 g in 20mL deionized water,82°C water bath, and gradually added NaCS to make it more dense and more viscous. It was really a time-consuming process to Dissolve NaCS completely.

Time delay:
Goal: To do the ligation of luxI-TT and luxR-TT. Analysis TT, luxpR, luxR, luxI
Isolate plasmids and ran on a gel. The result for ligation was not good for the bands of TT were not clear. Change the ligation system: adding 2µL digested vector-TT and 6µL digested insert-luxI and luxR.
The result for ligation was not good for there are four bands of luxI and luxR, both two of them are correct. So we extracted gel and did gel ligation and put it in 4°C, waiting for transformation.
The transformation of RBS-GFP-TT was failed.

Team Meeting:
Sophia presented experiments progress of cell digital display and plan for this week: to finish construction of all the devices. Qiu introduced their ideas and project for time-delay display. Hu made a brief review of principles in cell immobilization. As for the method, they preferred sodium alginate(SA), which is natural and less toxic to cells. Moson gave an brief introduction about recent progress and remained issued, such as exhibition venue, typesetting, potential sponsors.

Hu: immobilized cell
Introduced principles and some basic knowledge of cell immobilization.
SA(natural but less strong) 3%

Moson XIE

• Recent Process: Resource almost prepared, planning document, background designed, price survey.
• Issue remained: exhibition venue (Aug 23-27th), Typesetting(Aug 10), background to be selected, potential sponsors.

Que Huang:
She presented several edition of logo and team name. Still many works left for design group. Wu asked them to finish design of logo and name in several days.

Tuesday, August 7th
Experiments:
Digital Display:
Today was a TERRIBLE day for experiments. After restriction analysis, all bands seemed to be correct, which cheered up everyone. However, another gel for ligation was abysmal. Six bands were observed after double digestion! Maybe enzyme was contaminated or added wrongly. Only thing we could do was to start all over again.

Cell Immobilization:
Got approximate 5% NaCS solution. Put the 6% PDMDAAC solution on a magnetic stirrer and stirred it at a certain speed, maintaining a small eddy in the center of liquid surface. Then dropped the NaCS solution into the fringe of the eddy by a 1mL injector. The drop then formed a spheroidic membrane. And if the rotating speed was too high, that spheroidicity will be torn and destroyed. The denser the NaCS solution is, the harder the microcapsules are destroyed.
After testing, we decided to dissolve more NaCS, until approximate 7%.

Time delay:
Goal: To do the ligation of luxpR-RBS-GFP-TT and reanalysis luxI and luxR.
Isolate plasmids and ran on a gel. The result for luxI is good, but had some bands behind the correct band of luxR in the single digestion analysis.
The result for ligation was not good for gel extract.

Design:
Brainstorming based on ideas we had in weekly meeting. The team’s name was mainly discussed. Several new ideas had been the focus. And the one, E.lomoile, became the final team name.

Wednesday, August 8th
Digital display:
Ligate Part II-III-IV and V-IV, I and II-III-IV, V and I, V-II and III-IV-I-IV.

Time delay: ligate luxPR and RGT
Today was quite BUSY! The number of tubes prepared for plasmid isolation raised up to 24! We had to divided them into two groups and perform isolation separately. Gel for restriction analysis was a disaster! Only one sample seemed to be right, whereas others were terribly wrong. Maybe the workload was too heavy to prevent any mistake, or maybe the bacteria were so naughty not cooperating with us. Wu advised us to decrease workload and perform ligation in a higher quality standard. In the evening, Hu wanted to do another restriction analysis using the same bacteria as morning's. We REALLY want to know why gel looked like that. Fortunately, the bands were closer to Mr. Right, though several wrong bands could be observed. Wang decided to give up glycerol stocks and reduce the workload, hoping to make all things correct.

Cell Immobilization:
After getting the NaCS solution in a proper concentration, we started to make the microcapsules entrapping engineering bacteria with plasmid PcI-RLT.
Centrifuged 10mL sample and collected the deposit. Added 10mL NaCS solution and mixed it thoroughly with the cells. Put the 6% PDMDAAC solution on a magnetic stirrer and stirred it at a certain speed, maintaining a small eddy in the center of liquid surface. Then dropped the mixture into the fringe of the eddy by a 1mL injector until it formed a spheroidic membrane. It took 10 min to react and completely form microcapsules. After that, we tipped all microcapsules to a strainer and rinsed it with sterile water. Then transferred all microcapsules  into  LB medium with Ampicillin, 37°C shaker incubated at 100rpm.
The new-made microcapsules were filled and translucent. But after 4 hours the culture solution appeared turbid, showing that there were some microcapsules broke and bacteria leaked into the medium.

Time delay:
Goal: To do the ligation of luxpR-RBS-GFP-TT and analysis luxpR and luxR.
Isolate plasmids and ran on a gel. The result of analysis for luxpR and luxR is wrong.
Reanalysis 2M19,the result is correct.
The transformation of luxI-TT and luxR-TT were successful.

Design:
We had a meeting to discuss the logo and designed several new versions.

 Thursday, August 9th
Experiments:
Digital Display:
Transferred the ligation product of the previous day and spread it. Isolated plasmid in order to ligate with product from gel extraction of the previous day. All things ran well, which made us feel extremely relived. Also picked colonies of plates of August 5th, and isolated plasmid for restriction analysis. Gel was perfect, clear bands and no other unwanted thing. So the rest of plasmids were available for ligation in the next day.
In order to verify all the glycerol stocks of PBAD and PBAD-RBS-cI-TT, added 100uL aliquot to LB medium separately and shake in 37°C.

Time delay:
Goal: To do the ligation of RBS1.0-luxI-TT, luxpR-RBS-GFP-TT.
Isolate plasmids and ran on a gel. The analysis of RBS1.0, 0.6, 0.3, 0.07, luxI-TT, 2M19 were correct so we extracted RBS1.0 luxI-TT, 2M19 to do the ligation of RBS1.0-luxI-TT, luxpR-RBS-GFP-TT.

Design:
We decided the formal one of the logo.

Friday, August 10th
Experiments:
Digital Display:
Isolated plasmid and digested for restriction analysis. Also digest the plasmid for ligation, which verified in the previous day. In the afternoon, almost every member went to a presentation delivered by Professors from Taiwan. Before we left, Wu set up a program which supposed to automatically shut down the electrophoresis apparatus after 30 minutes.  However, it did not work!! Gel kept running for 48 minutes until Ma figured out something wrong. We came back, nervous and regretful. Anyway, brought gel to take a photo. Fortunately, it seemed that Ma's timely pause prevented a disaster: the bands were just around the border. PBAD was correct, another gel was extracted for ligation. With great regret, Wu decided to isolate plasmids personally and ran another gel to get better a electrophoresis photo.

Cell Immobilization:
Goal: To determine a PcI-RLT standard curve.
1000mL LB medium with 1 mLAmp+ 55 mL PcI-RLT aliquot.
From 0 h on, took and collected 1mL sample in Eppendorf tube per hour, storing at 4°C. Repeatedly operated until the 12th hour. Determined each sample's OD value.
From 4 h on, took 10mL sample, centrifuged for 3min at 6000 rpm and collected the deposit cells, storing at 4°C.Repeatedly operated until the 10th hour. 65°C drying to completely dry,  then measured each sample's dry weight.
The OD curve seemed to be normal, but the dry weight and OD values were not showing an obvious linear relationship. Through analysis, we considered the error might be brought in the drying process. We didn't have all samples drying for an equivalent period of time.

Time delay:
Goal: To do the ligation of RBS0.6-luxI-TT, RBS0.07-luxI-TT, RBS0.3-luxI-TT
Isolate plasmids and ran on a gel. Cause we had analysis RBS0.6, RBS0.3, RBS0.07 and luxI-TT yesterday and the result for ligation was good, we extracted gel and did the ligation.
Isolate plasmids and ran on a gel for next day as well.RBS0.01 and luxR-TT was right.

Design:
We met for uniform design and got preliminary schemes.

 

Saturday, August 11th
Experiments:
Digital Display:
Continue the construction, Isolation, digestion and ligation. Also made 8 tubes of competence BL21.

Cell Immobilization:
Prepared calcium alginate and embed engineering bacteria with plasmid PcI-RLT.
PcI-RLT beads emitted fluorescence in CaCl2 solution.
Prepared NaCS solution (0.7706g in 15mL deionized water) and tested its concentration.
Result: The concentration was too low so that the spheroidic membranes were easily torn. So we decided to dissolve more NaCS.

Time delay:
Goal: To do the ligation of RBS0.01-luxI-TT, RBS1.0-luxI-TT, and do the ligaion of RBS1.0-luxR-TT again.
Isolate plasmids and ran on a gel, RBS1.0 was correct but little. LuxR-TT and luxpR2M19 were correct. The target gene of luxI-TT was too little to be seen, we did not extract it.
The transformation of RBS1.0-luxI-TT was failed but luxpR-RBS-GFP-TT was correct.

Design:
We finished the first draft.

Sunday, August 12th
Experiments:
Digital Display:
Today went smoothly, except for the plates of the previous day. We cannot figure out why no colonies could be observed in some plates, while others grew well. Competence cells were made in the same time. Wang looked up all the possible reasons and was perturbed about the competence cells she made and transformation procedure. To find where the problem is, she did a positive control using the remained competence cells.  If the positive control does not produce the expected result, there may be something wrong with the experimental procedure, and the experiment is repeated.

Cell Immobilization:
Goal: To prepare NaCS.
Result: After 66min's reaction, we didn't completely squeeze out all the reaction liquid from the reacted linters, therefore the subsequent processes were impeded. At last we didn't get proper production.

Time delay:
Goal: To do the ligation of RBS0.3-luxI-TT.
Isolate plasmids and ran on a gel. The transformation of RBS1.0-luxI-TT, RBS0.6-luxI-TT, RBS0.3-luxI-TT, RBS0.07-luxI-TT, RBS0.01-luxI-TT and RBS1.0-luxR-TT were failed before,  we do the ligation again. Cause RBS0.3 was stored at -20℃, we only isolate plasmids of luxI-TT.
The transformation of RBS0.6-luxI-TT, RBS0.07-luxI-TT and RBS0.3-luxI-TT were failed.

Design:
Other drafts were finished. Other details of the uniform had been sure.

 

 Week 19: August 13th to 20th
Monday, August 13th
Experiments:
Digital Display:
Another sad day for the lab. No colonies can be observed in the transformation plate except for positive control. If the problem was not competence BL21 or transformation process, ligation is to blame for the failure. For another part, isolated plasmid and digest for restriction analysis. The electrophoresis photo indicated the failure of ligation. Since the part get longer, there is a great chance of unsuccessful ligation.
Wang found water has leaked into some plates preserved close to the wall of the 4°C refrigerator. It seemed that parafilm was not so airtight and waterproof as we thought. She took out them and streaked the plate once again.

Time-delay:
Goal: To do the ligation of PBAD-RBS-luxI-TT and PBAD-RBS-luxR-TT and analyze luxpR-2M19.
Isolate plasmids and ran on a gel. The result for analysis was correct but not fully cut. As for extraction, the bands of luxpR-2M19 were correct and clear to be extracted.
The transformation of RBS0.01-luxI-TT, RBS1.0-luxI-TT, RBS1.0-luxR-TT and RBS0.3-luxI-TT were failed.

Cell Immobilization:
Goal: To prepare NaCS.
Result: After adding alcohol, some white floccule appeared on the top of the solution, and it gradually separated to  several layers. The upper layer was retiary floccule, the second layer was transparent liquid, and the third layer appeared to be cloud form floccule (It might be production whose chains were shorter than normal length).  Centrifuged and collected the upper retiary floccule, 65°C drying.

Design:
We decided the uniform design in the weekly full meeting and discussed the wiki design proposals.

Team meeting:
Sophia presented the experimental progress. Have finished all the parts and some ligation between parts. Fluorescence test would be determined by SpectraMax M5 Multi-Mode Microplate Readers(Molecular Devices). She reiterated that group leaders should send her daily report in time.
Shuqin introduced their work on cell immobilization last week. Two method, calcium alginate and NACS PDNDAAC microcapsule, were performed at the same time. They will finish CAD drawing of equipment next week. Yunxin presented the journal her wrote about their group. She also referred paper research on unstable GFP. Wu thought more research were needed, especially on those iGEM teams who had experience on unstable GFP. It takes about one week to prepare the NACS. Wu pointed out that we should succeed in cell immobilization first, then to consider improvement methods. Some preparation tests will help the next stage works.
Muxin showed her works on team description. Some members still haven’t handed in their personal description. Moson delivered his presentation in English. Maybe we all should try this next time. Youbin mentioned about his ideas about fluorescence test. Rong presented achievement on design. T-shirt template have been finished. Design on wiki still keeps on going.

Tuesday, August 14th
Experiments:
Digital Display:
Ligated circuit B in 16°C for another try using the remained ligation product. After 4 hours, transferred into competence BL21 and spread on plates. In case of the second failure of ligation, incubated genetic parts for another isolation & ligation cycle. To eliminate interference from competence cell, positive and negative controls were conducted at the same time.

Time-delay:
Goal: To do the ligation of luxR-TT-luxpR-RBS-GFP-TT.
Isolate plasmids and ran on a gel. The result for analysis was correct and for ligation was good, we extracted gel and did gel ligation and put it in 4°C, waiting for transformation.
The ligation of PBAD-RBS-luxI-TT and PBAD-RBS-luxR-TT were failed.

Cell Immobilization:
Goal: To determine a PBAD-RLT standard curve.
400mL LB medium with 400uLAmp+ 2mL PBAD-RLT aliquot , started at 8:56.
From 0 h on, took and collected 1mL sample in Eppendorf tube per hour, storing at 4°C. Repeatedly operated until the 13th hour. Determined each sample's OD value.
From 4 h on, took 10mL sample, centrifuged for 3min at 6000 rpm and collected the deposit cells, storing at 4°C.Repeatedly operated until the 13th hour. 65°C drying for 12 hours and then measured each sample's dry weight.

The OD curve was not a smooth S-Curve. The sampling process might bring some error to the determination.
Casting out the last three interferential OD values, the association degree of standard curve  is 0.92.

Design:
We engaged in Wiki design and finished the drafts. 

Wednesday, August 15th
Experiments:
Digital Display:
Still no colonies on the transformation plate and control groups all behaved in a normal manner. Maybe ligase or buffer was contaminated or became inactive. We changed ligase and buffer and hope for the best. Put the mixture in 4°C for 16 hours.

Time-delay:
Goal: To do the ligation of RBS0.01-luxI-TT, RBS0.07-luxI-TT, RBS0.3-luxI-TT, RBS0.6-luxI-TT.
Isolate plasmids and ran on a gel. The result for analysis was correct and for ligation was good, we extracted gel and did gel ligation and put it in 4°C, waiting for transformation.

Cell Immobilization:
Goal: To prepare NaCS.
Result: After adding alcohol, some white retiary floccule appeared on the top of the solution. Centrifuged and collected the upper floccule, 65°C drying.
Prepared calcium alginate and embed engineering bacteria with plasmid PBAD-RLT.
The NaCS production made on August 13th presented to be tawny, and dissolved more rapidly, But the solution was less stringy than it should be. So we judged the production was impure.

Device construction:
Prepared 7 glass tubes (inner diameter 10mm) with rubber plugs, 14 glass adapters (inner diameter 5mm) and an Expanded Polystyrene board. Perforate the board and roughly established our device.

 

Thursday, August 16th
Digital Display:
Transferred the ligation products and spread them on the plates. To find out where the problem is, Wang decided to conduct another two controlled experiments to verify two things: completeness of double digestion of backbones, efficiency of competence BL21.

Time-delay:
Goal: To do the preparation for ligation of RBS_0.01-luxI-TT, RBS_0.07-luxI-TT, RBS0.3-luxI-TT,
Isolate plasmids of luxI-TT and luxR-TT. Do the ligation of RBS_0.01-luxI-TT, RBS_0.07-luxI-TT, RBS_0.3-luxI-TT and did the transformation 4 hours later.

Cell Immobilization:
Transferred the PBAD-RLT  beads into  20mL LB medium with Ampicillin, 37°C shaker incubated. However the quantity of PBAD-RLT beads was too small, and they were not of uniform size, so we abandon them.
Again, prepared calcium alginate and embed engineering bacteria with plasmid PBAD-RLT.

 

Friday, August 17th
Digital Display:
In the morning, we sensed different when coming into the lab: an air of colonies!!! Though there were only a part of the plates with growing colonies, we still felt inspired. Picked one colonies of each plates into LB medium and put in the shaker for the verification this night. Also streaked plates using several randomly selected colonies.
Terrible nightmare in the evening: colonies we picked all turned out to be false-positive. We had no idea where the problem was.

Time-delay:
Goal: To do the analysis of luxR-luxpR-2M19 and RBS_0.01-luxI-TT and do the ligation of P_BAD-RBS-luxR-luxpR-2M19.
Isolate plasmids and ran on a gel. The result for analysis of luxR-luxpR-2M19 was wrong, so we did not extract gel. And the result for ligation of luxR-TT and luxpR-2M19 was clear and correct so we extracted gel and did gel ligation and put it in 16℃, did the transformation 4 hours later.
The ligation of luxRTT-luxpR2M19 was wrong.

Cell Immobilization:
Transferred the PBAD-RLT  beads into  20mL LB medium with Ampicillin, and added 2mL Ara 37°C shaker ( 50r/min)incubated.
Prepared NaCS, 65°C drying.
Prepared calcium alginate and embed engineering bacteria with plasmid PcI-RLT.

Design:
We confirmed the frame, color scheme and more details of the Wiki and engaged completing the former scheme. The arrangement of the homepage was changed to be more simple and clear. A photo gallery was made.

 

Saturday, August 18th
Digital Display:
Another awful day: We were deeply suspicious of ligation process, especially of ligase and buffer. So we lent these from lab 571 and added into remained gel extraction product. Wu still clung to the hope that the plate with colonies could not all be false-positive - they had probability to be right. He picked ten colonies into LB medium and wished for the best.

Time-delay:
Goal: To do the analysis of RBS_0.3-LuxI-TT.
Isolate plasmids and ran on a gel. The result for analysis of RBS0.3-luxI-TT was wrong.
Did the ligation of and luxR-TT to test the T4 DNA ligation liquid cause luxR-TT had been successful linked before.

Cell Immobilization:
Filled the PcI-RLT beads in the glass tubes of our device, and tried to applied a fed-batch culture with a peristaltic pump.

Sunday, August 19th
Digital Display:
After isolation of plasmids and gel running, the electrophoresis showed that all the colonies picked from transformation plates were false-positive. It seemed that backbones haven't digested completely and ligated into circles again.  Therefore, maybe the problem is double digestion.

Time-delay:
Goal: To find the problem.
The ligations of luxR-TT, P_BADRBS-luxRTT and luxRTT-luxpR2M19 were failed.

Cell Immobilization:
Prepared calcium alginate and embed engineering bacteria with plasmid PcI-RLT (glycerin tube) and with PcI-RLT (single colony) .

Design:
We confirmed the frame, color scheme and more details of the Wiki and engaged completing the former scheme. The arrangement of the homepage was changed to be more simple and clear. A photo gallery was made.

 Week 20: August 20th to August 26th
Monday, August 20th
Digital Display:
Ligation again, using new endonuclease. Incubated another two parts, preparing for the ligation of the next day.

Cell Immobilization
Tested NaCS solution's concentration and prepared materials for our device construction.

Time Delay:
Goal: To do the ligation of RBS_0.6-luxITT.
Use the new T4 DNA ligase but the old Agarose Gel Extraction kit.
Isolate plasmids and ran on a gel. The result for analysis was not good because there were too many bands beside the correct bands. And the result for extraction was clear and good so we did the gel extract and did the ligation of RBS0.6-luxITT and put it in 16°C.

Design:
We discussed all work of last week and made a plan of next week. Several principles were emphasized again.
  
Tuesday, August 21st
Digital Display:
Another ligation. Transferred ligation product of the previous day.

Cell Immobilization:
Experiment:
Goal: To prepare NaCS.
Result: After adding alcoholm, some white retiary floccule appeared on the top of the solution.Centrifuged and collected the upper floccule, 65°C drying.

Time Delay:
Goal: To do the ligation of PBADRBS-luxITT and P_BADRBS-luxRTT
Transform the ligation of RBS0.6-luxITT and isolate plasmids of luxITT and luxRTT. Cause cells with PBADRBS wasn’t much enough to be isolate, we just did gel extract of luxITT and luxRTT. The bands of luxITT and luxRTT were correct and clear. Put them in 16°C.

Wednesday, August 22nd
Digital Display:
A graduate, researching in molecular bio in Lab 571, has conducted ligation successfully in the previous day. To verify whether the isolation kit or other factors in our lab were alright, we divided into two groups and conduct experiments in both lab 580 and 571. Misfortunes never come singly: electrophoresis photo indicated that isolation, which had never caused any problem before, all turned to be failed!

Time Delay:
Goal: To do the ligation of  RBS_0.01-LuxITT, P_BADRBS-luxITT and P_BADRBS-LuxRTT.
Use the new High Purity Plasmid Miniprep Kit.
Isolate plasmids and ran on a gel. The result for analysis of RBS0.01 was correct so did the gel extract and did the ligation of RBS0.01-luxITT, P_BADRBS-luxITT and PBADRBS-luxRTT. Did two ligations of PBADRBS-luxRTT, one is put in 4°C and the other is in 16°C to test the effect of ligase.

Fluorescence Test:
Fluorescence measurements were designed to determine the top values of fluorescence and the time when the value of fluorescence fall. We set a group of inducers included four high levels of arabinose concentration in today’s fluorescence measurements of PBADRLT. Measurements were taken in a time interval of 15 minutes. We got a series of data containing 18 groups of value in every level. These data was enough for us to get four curves of fluorescence.

Thursday, August 23rd
Digital Display:
Today is the Chinese traditional Valentine's Day, but we had no mood for it.
Original plan is to perform the controlled experiments again to verify the ligation procedure is alright. Bad things happened again: no bands in electrophoresis photo. Maybe the plasmid isolation kits were contaminated or something wrong with purification columns. The band of RETPcIRLT seemed strange, maybe need another ligation.
Made competence DH5α. Wang changed the rotational speed from 6000rmp to 4000rpm. Then transferred the ligation products of the previous day and spread plates.

Cell Immobilization:
Experiment:
Prepared calcium alginate and embed engineering bacteria with plasmid P_BAD-RLT.
Prepared NaCS, 65°C drying.

Time Delay:
Goal: To do the ligation of  RBS_0.01-luxITT, P_BADRBS-luxITT, P_BADRBS-luxRTT and luxpR-2M19.
Transform the plasmids of  RBS_0.01-luxITT, PBADRBS-luxITT and PBADRBS-luxRTT into DH5α.
Isolate plasmids and ran on a gel. The bands could not be seen.

Friday, August 24th
Digital Display:
To ligate RET and PcIRLT once again, conducting controlled experiments to verify whether purification column is reliable. Unfortunately, it is solution that is making trouble, rather than columns. So we ended up with barely nothing. One shocking news happened in the late evening: verification of transformation plates all turned to be correct! Got so excited!

Cell Immobilization:
Experiment:
Rinsed P_BAD-RLT beads and transferred them into 20mL LB culture medium, adding 20uL Amp. 37°C shaker 50rpm incubated. After 2 hours, added 2mL Ara.
Put 5 P_BAD-RLT beads and 1mL culture fluid into an Eppendorf tube, storing at 4°C.
After adding Ara and incubating for 7h, 8h, put 5 P_BAD-RLT beads and 1mL culture fluid into an Eppendorf tube.
Determined each sample's fluorescence intensity.
Result:
0h sample's fluorescence intensity was the least, 8h>7h.
The fluorescence intensity is related to the amount of beads.
Because some of the bacteria leaked into the medium, the background fluorescence intensity brings a significant influence to the measuring.

Device construction:
Modified the device ( use double pipe inlets)

Time Delay:
Goal: To do the analysis of RBS_0.01-luxITT, P_BADRBS-luxITT, P_BADRBS-luxRTT.
Isolate plasmids and ran on a gel. The result for analysis was correct and clear.

Design:
We continually worked on building Wiki. Discussion on the scheme of the team part of the Wiki.

Saturday, August 25th
Digital Display:
To ligate RET and PcIRLT once again. Also isolated the plasmid of 18AI and RETPcPb for the ligation tomorrow.

Cell Immobilization:
Prepared calcium alginate and embed engineering bacteria with plasmid PBAD-RLT.
Debugged  two peristaltic pumps  for the numeric displayer device.

Time Delay:
Goal: To do the ligation of P_BADRBSluxITT- P_BADRBS-luxRTT, P_BAD- RBS0.01-luxITT and luxITT- P_BADRBS-luxRTT.
Isolate plasmids and ran on a gel. The analysis of P_BADRBS-luxR

Design:
We did some card design and drew some drafts.

Sunday, August 26th
Digital Display:
Ligated 18AI and RETPcPb and put the ligation product in 16°C overnight for the transformation in the next day.

Cell Immobilization:
Experiment:
Goal: To test the fluorescence intensity of P_BAD-RLT beads.
Transferred the PBAD-RLT  beads into  20mL LB medium with Ampicillin, 37°C shaker ( 50r/min)incubated.
After 2 hours, added 2mL Ara. Took 5 PBAD-RLT beads and rinsed them with PBS, then Started  fluorescence test (exciting laser intensity: 485 nm, emitting laser intensity: 520 nm). Tested on 0.5h, 1h, 1.5h, 4h, 5h, 6h.
Problem: There was a little difficulty taking out the beads without breaking them, therefore the time we need to collect sample was prolonged.
When first taken out, P_BAD-RLT  beads emitted very low fluorescence. After a while in plates, the fluorescence became stronger.
Maybe the GFP proteins inside the bead emit fluorescence only after they are exposed to enough oxygen. When the beads are in the medium, the dissolved oxygen is relatively insufficient due to the low rotating speed.

Prepared a large number of blank calcium alginate beads.
Prepared calcium alginate and embed engineering bacteria with plasmid P_BAD-RLT.

Device construction:
Debugged the numeric displayer

Time Delay:
Goal: To do the ligation of P_BAD-RBS0.6 and P_BAD-RBS_0.07.
Isolate plasmids and ran on a gel. The result for analysis was correct. Cause the bands of P_BAD hardly to be seem, we increased the volume of P_BAD when ligation.

Fluorescence Test:
We changed the arabinose concentration from high levels to lower ones according to last fluorescence measurements of PBADRLT. Besides, we took some kinetic curves in all concentration levels we had taken before for comparison. The analysis of these data will help us improve our efficiency in the coming fluorescence measurements

Design:
We drew more drafts and talked them on the weekly full meeting.

September
Week 21: August 27th to September 2nd
Monday, August 27th
Digit Display:
Ligated RET and EPc again

Cell Immobilization:
Experiment:
Goal: To test the fluorescence intensity of P_BAD-RLT beads.
Each well contained 5 beads. Ara: 20 uL, 50 mM

As the graph shows, beads emitted approximate intensity of fluorescence when cultivated for 10h, no matter what time Ara was added. So we can add Ara after beads were transferred into culture medium for 3 h in our later experiments.

Device construction:
Filled the tubes with beads and tested the properties of tubes using a peristaltic pump to pump water through the tubes,
Finding: The flow of the pump was too small and the flow was blocked badly in tubes. Each tubes had different resistance to water, thus the flow in each tubes was different. While blasting air through tubes, an upright tube would   make air pass easier. Also, the flow should not be too fast, otherwise the beads will rush to one side and plug up the tube.

Team Meeting:
Sophia started meeting by her report on experimental progress in  digital display. Then she assigned the task of wiki construction-drafts writing! We need to

Tuesday, August 28th
Digit Display:
Continue the construction

Cell Immobilization:
Goal: To test the fluorescence intensity of P_BAD-RLT beads.
Each well contained 5 beads. Ara: 20 uL, 50 mM

As the graph shows, beads emitted approximate intensity of fluorescence when cultivated for 10h, no matter what time Ara was added. So we can add Ara after beads were transferred into culture medium for 3 h in our later experiments.

Device construction:
Filled the tubes with beads and tested the properties of tubes using a peristaltic pump to pump water through the tubes,
Finding: The flow of the pump was too small and the flow was blocked badly in tubes. Each tubes had different resistance to water, thus the flow in each tubes was different. While blasting air through tubes, an upright tube would   make air pass easier. Also, the flow should not be too fast, otherwise the beads will rush to one side and plug up the tube.

Fluorescence Test:
Since the analytical result from kinetic curves we had taken on last Sunday, we lowered the level of inducer concentration once again. But curves of fluorescence measurements in this time show something different from last ones. There are still remarkable fluctuations of parallel samples. These phenomena might result from the unstable and nonairtight environment in the SpectraMax M5 that obviously decreased the volume of Basal Medium.

Design:
Card design

Wednesday, August 29th
Digit Display:
Another construction day.

Cell Immobilization:
Experiment:
Filled a tube with beads, then pumped LB culture medium in and out of the tube with a peristaltic pump.
Prepared calcium alginate and embed engineering bacteria with plasmid PBAD-RLT, then put the beads into a black ELISA plate, Started cultivation and determination at 21:30 and ended at 9:30. (37°C, exiting laser intensity: 485nm, emitting laser intensity:520nm, Ara 50mM)
Wells:
A1-A5 200uL LB+ Amp                                 A6-A10 200uL LB+ Amp+ beads
B1-B5 200uL LB+Amp+beads+5uLAra      B6-B10 200uL LB+Amp+beads+10uLAra
C1-C5 200uL LB+Amp+beads+20uLAra    C6-C10 200uL LB+Amp+beads+20uLAra
D1-D5 beads+20uLAra                                D6-D10 200uL LB+Amp+20uLAra

There was no obvious difference in the intensity of fluorescence, despite different concentration gradients of Arabinose . The intensity of fluorescence that could be tested was very low.

 

The fluorescence increased at first and fade gradually. Maybe Ca-Alginate beads without LB medium can live and product green fluorescence protein in Arabinose.

Fluorescence Test:
 We took fluorescence measurements in a time interval of 15 minutes in the same gradient as last test. Although every curve still kept a good tendency, they showed that top values were totally low. We thought the new plate with week colonies should take the blame after a discussion. By now, on the other hand, we have gotten enough data so that we can determine the most suitable level of arabinose concentration.

Thursday, August 30th

Digit Display:
When Sophia add 100μL aliquot DH5α liquid culture to new LB medium, she also performed a negative control by adding same aliquot to LB with Amp. But after 1h45min incubation, the negative control turned opaque. So she had no choice but to give up newly made competence cells, using the one made two weeks ago to instead, which preserved in -80ºC. We held little hope for this transformation.

Cell Immobilization:
Experiment:
Goal:
1. To prepare intra-hollow Ca-alginate capsules.
2. To prepare blank capsules: SA: 1%, CaCl₂: 10%, CMC: 1.2%. 10mL CaCl₂ +10mL CMC,  mixing thoroughly. Drew the mixture in a 10mL injector, then dropped it one by one through the pinhead into 100mL  SA solution, gel for 10 min. Then rinsed the capsules.
3. To prepare  intra-hollow capsules: SA: 1%, CaCl₂: 2%, CMC: 1.2%. 1mL sterile water + 5mL CaCl₂ + CMC mixture, mixing thoroughly. Drew the  mixture in a 10mL injector, then dropped it one by one through the pinhead into 100mL  SA solution, gel for 5 min. Then rinsed the capsules and put them into 3% CaCl2 solution, stirring for 20 min.
Prepared calcium alginate and embed engineering bacteria with plasmid PBAD-RLT. Modified the ratio of cells and sterile water to 1:5.

Design:
The design of the card is undergoing extensive modification.

Friday, August 31st
Advisor Wu and Sophia will be away for a few days, so Wu changed our team meeting from next Monday to this Friday.
Team Meeting:
As decided this Monday, we presented our report in English. Sophia spoke first, as usual. Then Xinyi, at the first time, delivered his speed in front of us.

Digit Display:
Ligation for construction of cell (a,d).

Cell Immobilization:
Experiment:
Embedded PcI-RLT and P_BAD-RLT in intra-hollow Ca-alginate capsules. Preserved them at 4℃.·  
2.5mL1.2%CMC+2.5mL2%CaCl2+1mL cell suspension dropped into 100mL1% alginate solution ·
Goal: Fluorescence test of PBAD-RLT beads:· 
Transferred Ca-alginate beads into 20mL LB medium with 20uL Amp, then added 200uL 500mM Arabinose aqua. Picked up 5 beads into a 1.5mL Centrifuge Tube immediately. Eluted the beads with 200uL PBS buffer solution. Then put the beads into a well of a black ELISA plate to test their fluorescence. All steps were performed under aseptic conditions.·  
After 1h, picked up another 5 beads to test their fluorescence·  
Test fluorescence from the first hour to the tenth hour.

The intensity of fluorescence increased in the first 5 hours and the maximum reached about 20,000. Then it decreased to 3,457.9. But the intensity of fluorescence had a tiny rise at the tenth hour. Possible reason was that the bacteria was starting a new growth cycle. As we didn't continued to measure the fluorescence after tenth hour, it is difficult to explain the result. So we will repeat the experiment again later.     

 

Saturday, September 1st
Digit Display:
 Still encountered the same problems on circuits.

Cell Immobilization:
Experiment:
Prepared intra-hollow Ca-alginate capsules with plasmid PBAD-RLT and PcI-RLT separately.
Preserved at 4 degree centigrade in sterilized distilled water.
Results: The capsules were not spherical and some droplets stuck together.

Sunday, September 2nd
Digit Display:
After discussion with Mrs. Wu, we thought that it is inappropriate to use BL21 during the whole construction process. Strains should first be in DH5α and assemble together.

Cell Immobilization:
Experiment:
Transferred the PBAD-RLT and PcI-RLT capsules into 20mL LB medium separately and cultivated them for 12 hours. Put 200uL 500mM Arabinose solution into the PBAD-RLT culture medium  at the second hour. 
Results:  Both capsules embedding the bacteria with PBAD-RLT  and with PcI-RLT were not fluorescent, and the culture medium were turbid, telling that some bacteria had leaked from the capsules.

Prepared intra-hollow Ca-alginate capsules with E.coli with plasmid PBAD-RLT .
Results: The capsules were trailing, not as spherical as we supposed them to be. After observation and analysis, we considered that the 1.2% w/v CMC solution and 2% w/v CaCl₂ solution mixture had changed its structure after autoclaving, and the viscosity and density of the cationic mixture had decreased. So the way of sterilization should be modified.

Fluorescence Test:
We chose certain levels of Arabinose concentration after analyzing the data we had gotten from experiments before to set a new gradient. In this time, we used a stronger colony from a new plate with revived strain. As we expected, fluorescence intensity of all samples kept at higher levels than ones of experiment on Wednesday. Both top value and the time when it appears of every sample with specific arabinose concentration are almost parallel with previous ones. Besides, we also used a new type of clear plate with a cap to take kinetic fluorescence measurements with the same gradient of arabinose concentration after we finished interval test.  But curves showed us an unsatisfying result – top values were totally low and tendencies of fluorescence intensity were not obvious. We thought the cap of the plate made the strain grew in an oxygen-limited environment so that green fluorescence proteins could not be oxided So far, tests about fluorescence intensity of the strain with a plasmid called PBADRLT have nearly been finished.

Design:
We came up with an idea about the mascot race and made a picture to illustrate it.

Week 22: Sept 3rd to Sept 9th
Monday, September 3rd
Digit Display:
To verified whether cell(a,d) is successfully constructed, we picked one colony from transformation plate and isolate plasmids. After digestion and electrophoresis, we found the ligation was failed, so ligation was performed again.

Cell Immobilization:
Experiment:
Prepared  intra-hollow Ca-alginate capsules with distilled water inside.
Tried to make capsules out of super clean bench. Prepared the cationic solution and the sodium alginate solution without sterilization, but kept the solution at the same ratio. Cell suspension was replaced by equal distilled water. Other steps remain as usual.
Results: The capsules we made were in perfect round shape, when using the solutions without autoclaving. Comparing the recent two batches of capsules, new methods of preparing and sterilization of solutions should be applied.

Fluorescence Test:
As a new circuit finished by the group of Time Delay, tests about it were taken today. We referred to the data we have gotten before for these tests, since this circuit also includes the PBAD – a promoter which we had also used as a brick to structure the PBADRLT. We made a mistake that we used the DH5α, which is good at gene constructions but not protein expressions, instead of BL21 so that protein expressions of this strain got low levels.

Design:
We complete the files of the card print and printed some samples of it.

We took attempts of the demo video and discussed more details.
The material for costume of mascot race was prepared.

Tuesday, September 4th

Cell Immobilization:
Experiment:
Prepared  intra-hollow Ca-alginate capsules with E.coli with plasmid PBAD-RLT .
Placed 10mL 4% w/v CaCl₂ solution and 2.4g CMC powder into two different 200mL beakers. Prepared 100mL 1% w/v sodium alginate solution .
Sterilized the two beakers (contained CaCl2 solution and CMC powder respectively) and the alginate solution in an autoclave at 120kPa and 121℃for 20 min.
After autoclaving, dissolved the CMC powder with 10mL distilled water and mixed with 10mL 4% w/v CaCl2 solution.
Mixed 1.5mL 4% w/v CaCl2 solution and 1.5mL CMC solution with 0.6mL cell suspension, then dropped the mixture into vortical sodium alginate solution. 
Results: The droplets also couldn't be a normal sphere. However, when the mixture of the same ratio was dropped into 1% w/v alginate solution without autoclaving, the droplets could form round and transparent capsules.  Even the same cationic solution with autoclaving still could produce normal spherical capsules. Therefore, we judged that autoclaving had a significant impact on sodium alginate solution. The alginate solution through autoclaving became pale yellow and its viscosity decreased.

Wednesday, September 5th

Digit Display:
Big day for experiment, not a good big however. When discussing our circuits with Xuesong, we found there might be some problem in construction work. Therefore, we asked Mrs. Wu's advices. She suggested to use DH5α rather than BL21 as competence cells and to perform ligation again from parts. BL21 is not a safe choose for storing plasmids, which is a fact that we were too late to realize. That means we are going to have a tough time, much tougher than before.

Cell Immobilization:
Experiment:
Prepared intra-hollow Ca-alginate capsules with plasmid PcI-RLT.
Result: The capsules were spherical and droplets rarely stuck together. But they didn't emit fluorescence as well as they were supposed to. Maybe the bacteria sample didn't carry a steady PcI-RLT and some of the plasmid had lost during the transfer process. Or we didn't effectively embed the bacteria into the capsules.

Fluorescence Test:
While we were preparing the fluorescence measurements this morning, we found strain – PBADRETPcIRLT4J – falsely expressed in the basal medium. So we canceled today’s test.

Thursday, September 6th

Digit Display:
Sophia flight from Kunming this morning and back to work.

Cell Immobilization:
Experiment:
Prepared intra-hollow Ca-alginate capsules with plasmid PBAD-RLT. After cultivated in 100rpm shaker for 3 hours, added 500uL 5mM Ara.
Result: The capsules were almost spherical, but some were trailing, and droplets rarely stuck together. But after adding Ara, they didn't emit fluorescence in the following 8 hours. The reason is still being explored.

 

Friday, September 7th

Digit Display:
Isolate plasmid and ran on a gel for restriction analysis. Result was still disappointing. We now gave up the original plan completely and construct circuits from parts. Sophia had transformed some plasmid to DH5α before, so we felt a little release.

Cell Immobilization:
Explored a better procedure, and modified the ratio of the solutions.
Prepared more spherical capsules which seldom leak.

Fluorescence Test:
We took a long test from yesterday afternoon to this morning for determining the fluorescence intensity curves of a strain constructed by the group of Time Delay. And we designed a gradient with four levels of arabinose concentration – 0, 0.1, 1, 10 mM. Data from this test would be references for our next test about other homogeneous strains. In general, data gave no distinct connection between arabinose concentration and values of fluorescence intensity. And curves pointed that we could use a lower level of arabinose concentration in next tests.

 

Saturday, September 8th

Digit Display:
Four transformation plates

Cell Immobilization:
Prepared intra-hollow Ca-alginate capsules with plasmid PBAD-RLT.
Observed the intra-hollow Ca-alginate capsules and measured their sizes under a microscope.

 

 

 

Sunday, September 9th

Digital Display:
Transferred parts into DH5α.

Cell Immobilization:
Experiment:
Prepared intra-hollow Ca-alginate capsules , which embedded 0.6mL PBAD-RLT cells suspension.
Fluorescence test of PBAD-RLT: No apparent fluorescence could be detected.

Fluorescence Test:
Since our Molecular Device only can measure the OD600 in limited range, we changed our protocol of fluorescence measurements as we changed the inducing time to 3 hour after diluting the overnight culture in new medium. From now on, samples taken from culture should be diluted to 4 times in PBS. Besides, we try to use the quotient of fluorescence intensity divided by OD600 as Y axis in graphs. So, we take a fluorescence measurement of the strain called PBADRLT on this new way today. To our delight, we got a good result.

 Week 23: Sept 10th to Sept 16th
Monday, Sept 10th

Cell Immobilization:
Prepared intra-hollow Ca-alginate capsules, embedding condensed cell suspension with plasmid PBAD-RLT. Measured the fluorescence of the capsules.

Fluorescence Test:
Some circuits had been finished by group of Time delay. So, we took two circuits of them to take fluorescence measurements so that we could make some comparisons between them. This time, we took long-time measurements to get enough data and graphs also showed us an expected result.

Tuesday, Sept 11th

Digital Display:
No colonies can be observed on the transformation plates, so did the positive control. The activity of BL21 might decrease. Did another ligation and looking for new BL21 at the same time.

Cell Immobilization:
Prepared intra-hollow Ca-alginate capsules at night, embedding condensed cell suspension with plasmid PBAD-RLT.  After transferring the capsules to LB culture medium, kept stationary culture for 10 hours.

Wednesday, Sept 12th

Digital Display:
NO COLONIES! Why? We gradually realized that molecular biology often have no why: it happen without reason. Only thing we can do is do it again, more carefully and cautiously. Sophia made 12 tubes of competence BL21 and DH5α, for complete circuits and ligation product respectively.

Cell Immobilization:
Measured the fluorescence intensity of capsules prepared at last night.
Prepared intra-hollow Ca-alginate capsules at night, embedding condensed cell suspension with plasmid PBAD-RLT. 
After transferring the capsules to LB culture medium, kept Shaken culture overnight.

Thursday, Sept 13th
Digital Display:
GREAT news today was that all transformation plates have grown colonies in them!! Though we cannot figure out why former ones have no colonies. Construction seems finally turning well, which made us feel so glad.

Plasmids of 4J from Kit Plate 2 transferred into BL21 earlier, so now we want to perform fluorescent characterize on it. First plasmids are isolated and digested for restriction analysis today. It turned out correct, so determination will conducted tomorrow.

Cell Immobilization:
Measured the fluorescence intensity of capsules prepared at last night.
Result: The fluorescence intensity was weak and instable, so we gave up after 6 hours.
Prepared intra-hollow Ca-alginate capsules, embedding condensed cell suspension with plasmid PBAD-RLT. 

Friday, Sept 14th

Digital Display:
Transferred the ligation product into DH5α.

Cell Immobilization:
Prepared intra-hollow Ca-alginate capsules, embedding condensed cell suspension with plasmid PBAD-RLT. Used a 1 mL syringe to extract a little culture inside a capsule, and observed it under a microscope. Meanwhile, spread some of the culture inside a capsule on an LB agar plate containing Amp, incubating overnight at 37°C .

 

On the next day, we found out that some bacterial colony had grown on the LB agar plate containing Amp, while there was no colony growing on the plate of control group.

Fluorescence Test:
Today is the first day when we tested the new circuit named 4J induced by aTc. But things got not well. According to the experiences, we made a gradient, 4 levels, of aTc concentrations – 0, 0.25, 2.5, 25 mM. All data gave us the only information that there were no green fluorescence protein expressions in our cultures. Answers to these questions are under researching and testing.

Saturday, Sept 15th
Digital Display:

Cell Immobilization:
Measured the fluorescence intensity of capsules prepared at last night.
The capsules were shaken for 10 hours after they were prepared. Then added Arabinose solution and measured the fluorescence of capsules.
Prepared intra-hollow Ca-alginate capsules with plasmid PBAD-RLT at night and transferred them to LB culture medium.

Fluorescence Test:
Since two new circuits of the group of Time Delay were constructed two days ago, we tested these circuits today with the same concentration of arabinose, 0.1 mM, as before. This time, we still wanted to get fluorescence / OD600 curves. But when we got results, we found that curves of these two circuits got coincident. It seemed that we might get some mistakes in our parts contribution.

 

Sunday, Sept 16th
Digit Display:
Still stick at the construction...

Cell Immobilization:
Measured the fluorescence of capsules which were prepared at a night before. After the capsules were prepared, they were shaken for 2 hours and kept stationary culture for 9 hours. Then added Arabinose solution and measured the fluorescence of capsules.

 

Fluorescence Test:
A protein electrophoresis about GFP-LVA was taken today. The photo accurately fit the fluorescence graph.

Week 24: Sept 17th to 24th
Monday, September 17th
Digital Display:
Transformation plates had colonies, which were then be picked into LB medium. However, the result was still disappointing.

Cell Immobilization:
Prepared intra-hollow Ca-alginate capsules with plasmid PBAD-RLT and transferred them to LB culture medium. After the capsules were prepared, they were shaken for 3 hours and then added Arabinose solution, measuring the fluorescence of capsules.
Team Meeting:
Sophia reported our terrible situation. We should have investigated more about promoter and its regulation principle. Now we put ourselves in an awkward situation, and we have no choice but to go ahead without hesitation.
Daniel showed us fluorescence test result. They assumed that TD0.07 might have been wrongly constructed.  The only good news was the successful of SDS-PAGE for PBADRLT.
Advisor Wu encouraged us, rearranging the schedule and hopping not to miss the deadline.

Tuesday, September 18th
Today is Wu's birthday, we had cakes and also forced

Digital Display:
Isolated plasmid of Ptet and RLT and performed electrophoresis. Then ligated the two parts. Since the unexpected result for the fluorescence test of 4J, ligation was conducted to improve the plasmid 4J, expressing more GFP(LVA) protein.

Cell Immobilization:
Prepared intra-hollow Ca-alginate capsules with distilled water. But the SA solution was not enough sticky due to being autoclaved for 20 min, and the capsule was not spherical.

Wednesday, September 19th

Digital Display:
Since new terms has begun this week, we gradually realized that we still have classes!!! In order not to miss the class, Sophia got up at 5 a.m. and made competence cells. Then transferred ligation production of the previous day. Xuesong helped us to spread plates. Alex isolated plasmid and Ruosang ran the gel. Another tragedy is that our restriction endonuclease didn't work. Another additional experiment indicated that XbaI and PstI were the problem.
Transformation plates grew so slowly that we nearly thought we had failed again. But Xuesong told us to trust him and his great spreading skill.

Cell Immobilization:
Prepared intra-hollow Ca-alginate capsules with distilled water. Measured the size of beads and capsules with a microscope and calculated the average their diameters.
In general，the capsules are a bit bigger than the beads .
Observe beads and capsules with a microscope.

 

Thursday, September 20th

Digital Display:
Good news was that all plates had colonies! Ruosang picked them for the verify tonight. Xin wanted to prove his suspect about the leak of PBAD was correct, so we also performed SDS-PAGE and some other experiments. Alex, Qingshu and Xin stayed up late to verify the transformation result. All effort turned to be worthwhile when knowing that almost all bands in the electrophoresis image were correct. LB medium and plates were consuming in an incredible speed. Every day we  must prepare dozens of flasks. Xin get another glycerol stock of BL21 from his classmate and Sophia made competence BL21 and transferred the plasmid preparing for expression.

Cell Immobilization:
Measured the film thickness of intra-hollow Ca-alginate capsules( no bacterium inside) with microscope and calculated the average their diameters.

Fluorescence Test:
PcIRLTPBADRLT, one of our ternary parts, was taken the fluorescence measurements today. But, results showed a curve of this circuit without induction with an unexpected tendency. Maybe there were some mistakes we had taken. We will repeat this test in the future. Besides, test about 4J still gave us wrong results.

Friday, September 21st

Digital Display:
Alex isolated plasmids which is going to be transferred into BL21 and perform following characterization.

Cell Immobilization:
Measured the mechanical strength of capsules and beads using Testing Machine EZTest (SHIMADZU) . The test speed was 1mm/min. The mechanical strength of intra-hollow Ca-alginate capsules are stronger than calcium alginate beads.

 

Prepared calcium alginate and embed engineering bacteria with plasmid PBAD-RLT. After preparation, soaked the beads in 100 mL CaCl2 solution overnight.

Measured the fluorescence of PBAD-RLT capsules and PcI-RLT capsules respectively.

 

Fluorescence Test:
The circuits PBADRLT and PcIRLT were tested today. But after we diluted the samples, we got wrong data that kept a strange tendency. Curves of PcIRLTPBADRLT in DH5αstill got wrong tendencies. We must send it into BL21 and test it tomorrow.

Saturday, September 22nd

Digital Display:
Made competence BL21 and transfer the plasmid of the previous day.

Cell Immobilization:
Experiment:
Rinsed PBAD-RLT beads and transferred them into 2 bottles of 20mL LB culture medium with Amp. 37°C shaker 100rpm incubated. After 3 hours, added Ara and started measuring the fluorescence of the beads.

 

Prepared intra-hollow Ca-alginate capsules with plasmid PBAD-RLT and transferred them to 50 mL LB culture medium with Amp. After the capsules were prepared, they were shaken for 3 hours and then added Arabinose solution, measuring the fluorescence of capsules.

 

Prepared intra-hollow Ca-alginate capsules with plasmid PcI-RLT and transferred them to 50 mL LB culture medium with Amp. Right after preparation, started measuring the fluorescence of the capsules.

The E. coli grown weakly and the intensity of fluorescence of PBAD-RLT was very low.

Fluorescence Test:
It’s really a lucky day. We re-designed the method of fluorescence measurements for PBADRLT and PcIRLT. Fortunately, the result turned to normal direction. PcIRLTPBADRLT in BL21 finally gave us correct curves after the third try. In addition, we took all composite circuits of Time Delay for fluorescence test and we got a good result once again.

 

Sunday, September 23th

Cell Immobilization:
Experiment:
Prepared intra-hollow Ca-alginate capsules with plasmid PBAD-RLT and transferred them to 50 mL LB culture medium with Amp. After the capsules were prepared, they were shaken for 3 hours and then added Arabinose solution, measuring the fluorescence of capsules.
Prepared intra-hollow Ca-anbsp;100mL1%lginate capsules with plasmid PcI-RLT and transferred them to 50 mL LB culture medium with Amp. Right after preparation, started measuring the fluorescence of the capsules.

Fluorescence Test:
PtetRLT was send into BL21 yesterday. This circuit is different in RBS from the 4J. Today, we not only test its fluorescence but its protein electrophoresis. Two level of the aTc concentration were used. Results show this circuit working well. Besides, fluorescence measurements about PBADRETPcIRLT, one with induction and another without, showed no difference between them.
The experiment started from the evening and lasted to the next day. We stayed up late to get samples. Other team members prepared the coming presentation to Mrs. Wu and Mr. Fang.

Week 25: September 24th to 26th

Monday, September 24th

Cell Immobilization:
Experiment:
Goal: To measure the immobilizing capability of the beads/ intra-hollow capsules
Used a microplate reader to measure the OD value of the culture medium of the immobilized beads/ intra-hollow capsules.

From the result, we can find the OD value of the culture medium of beads grown up faster and higher, indicating that cells immobilized in them leak easier than those embedded in capsules.

Presentation:
Sophia, Daniel, Alex and Moson delivered the speech. Mrs. Wu, Mr. Fang, as well as other graduates have given some valuable advices.

Tuesday, September 25th
Wiki...wiki...wiki...

Wednesday, September 26th
Deadline...