Team:CSIA SouthKorea/Diary

From 2012hs.igem.org

Latest revision as of 01:22, 17 June 2012

Sep~Dec 2011

• 09.20.2011

-We didn’t know any professional knowledge about synthetic biology so we decided to ask for help of experts. While searching for the professors who participated in iGEM before, we found out that a professor from Korea University has participated several times before. So, we first sent an e-mail to our instructor In-Geol Choi to ask for his help.

• 09.21.2011

-The professor responded very quickly. And he was really positive toward us having interest in synthetic biology and willing to participate in iGEM.

• 12.18.2011

-While searching through the iGEM wikis, we got interested in making bacterial sunscreen, the idea from the imperial college 2011 wiki brainstorming section. Sunscreens that we normally use can only protect UVB, which is only one part of the broad range of UV. So, we were hoping to make bacteria that can protect UVB and UVA, another kind of UV light. And according to our further research about bacterial sunscreens, scytonemin, a pigment synthesized in cyanobacteria, absorbs UVA and pityriacitrin, a pigment synthesized in malassezia yeast, absorbs a wide range of UV. Thus, we thought we could use the genes that codes for this pigments and make a bacteria that carries the genes. However, we had so much difficulty finding it that we sent the second e-mail today for help from the professor.

• 12.19.2011

-We received an answer from our instructor. He said our idea was interesting and worth making it, but it would be too hard for us to make it as high school students. ☹ He also sent us a review related to bacterial sunscreens from the Nature: Microbial ultraviolet sunscreens by Qunjie Gao and Ferran Garcia-Pichel.

• 12.25.2011

-We sent our first e-mail to iGEM asking several questions about the competition.

• 12.28.2011

-We received a reply from the iGEM and she was very nice! ☺

Jan~Feb 2012

• 01.13.2012

-Today, we sent another e-mail to our instructor with several ideas. All of these ideas were from previous team wikis. From open wet ware, we found the rough outline of Stanford 2009 iGEM project. This team tried to degrade plastic, specifically phenol and formaldehyde, into carbon source of e.coli. However, we couldn’t find any sign that shows the team actually conducted the experimentation. Other one was making an e.coli that can sense methane. This idea was based on METU 2011 project. This team, had several projects-sensing methane, conversion, entrapment, killswitch- but they didn’t accomplish their goal. So we thought we could try part of their project, methane sensing. The last idea we sent was making e.coli that produces an apple smell. We found a similar project, MIT 2006 project, in biobuilder homepage that was to make a banana odor generator with e.coli.

-More information about our ideas can be found in brainstorming section of our wiki!

• 01.13.2012

-We received some negative comments on our ideas. He told us that since methane oxygenase is multimeric enzyme, we would have hard time sensing methane and also plastic degrading would be a tough task for us. But he said maybe using other kind of ester to produce odor could be possible. And he also suggested us to improve Korea University 2010 project.

• 02.16.2012: meeting with professor

-Our team had a meeting with professor In-Geol Choi in order to discuss our topic for the igem project. We had some creative ideas of our own, but we weren’t sure if they were appropriate topics for this project. As our discussion went on, professor Choi gave us some advices in selecting the topic:

-First, the project should have some kind of purpose – something like ‘saving environment’. We need to synthesize and create biological machine of certain function that can fulfill the fundamental purpose of our own.

- Second, considering that we don’t have much time nor the professional knowledge for the research and experiments, as high school students, it might be more appropriate to work with relatively easy project. He recommended us to do some research on websites such as biobuilders, and read about some labs that students can conduct.

-Third, note the parts registry that we can use. In parts registry site ( http://partsregistry.org/Catalog) we can find out which devices and functions we are able to use, and that will help us with selecting our topic. We decided to consider these points and brainstorm more about the topics. We are to determine our topic by the end of this February.

• 02.20.2012: ideas, ideas, ideas

-We first start to have an idea of making glowing bacteria to use it as a reading lamp. And having done some research, we found out that most of the glowing products were containing a protein called GFP (green fluorescent protein). To make a new challenge, we thought of making a new color of light other than green light. However, we soon found out that other great scientist have already made numerous mutation of GFP and produced many other colors. So, now… we are stuck!!!

• 02.17.2012

-After some discussions and research, we came up with some ideas: Lacotse intolerance curing ecoli, cholesterol degradation, and synthesis of scent. We referred to many of the previous igem projects and the research materials to determine necessary steps for each of the project.

-Lactose intolerance curing e.coli : mechanism of making E.Coli to do apoptosis when ther is excessive nutrient in human colon

It prevents excessive growth of E.Coli culture and reduces lactose-intolerant sypmtoms

1) Sensing lactose

2) Apoptosis signal

-because cell can’t die due to its own signal, genetically engineered E.Coli should be made resistant to apoptosis signal

-To prevent complete eradication of E.coli or too minimal deaths of E.Coli, the strength or period of signal should be controlled

-Cholesterol degradation

Bacteria called Pseudomonas sp. NCIB 10590 and Bcillus subtillis have the gene to degrade cholesterol. In human bodies, DHCR7 gene codes for enzyme 7-dehydrocholesterol reductase.

-Synthesis of scent using Candida rugose

(*openwetware)

Mar 2012

• 03.05.2012

-The idea of synthesis of scent

When we looked over the banana scent-synthesing project that MIT had conducted before, we found out that they applied arbitrary standard for the quantity of scent. But other than that, we couldn’t find a method of quantitatively measuring specific gas. We are still looking for mechanical sensors..

• 03.07.2012

We thought of additional ideas: killing mosquitoes and toggle switch.

-Killing mosquitoes

Mosquitoes have a preference for dextorotatory ocetenol molecules that are emitted y mammals.

During the research, we found out how to lure the mosquitoes and chemicals that can kill them. Engineering E.Coli that can synthesize both chemicals would be the goal.

-Toggle switch

Two positive feeback loop that represses each other

Idea 1) make an input that can sensor pH, and distribute in in the soil so that weak base can be released when needed

Idea 2) because toggle switch has similar mechanism with neuron, it might be able to model a simple neuron circuit.

• 03.12.2012

We received some negative feedbacks for our “cobalt buster” proposal. We made this proposal based on previous igem projects. However, he told us that it doesn’t seem to be able to be significantly improved, nor be more developed, since previous igem team had already completed much of the work. Instead, what we should focus on is developing the projects that prvious igem team had failed. What he advised was that because there is not enough time for highschool-division to go over all the errors and fix them, it would be better to plan a project with interesting ideas. He also suggested that in Cobalt Buster project, we can modify the operon so that we can create a circuit that has cobalt as the inducer.

• 03.13.2012

Professor Choi also sent us some feedbacks on our ideas. He first commented on our “mosquito-kiling” that it is a great idea, but we should consider more about the feasibility, and how long it would take to complete the project.

Also, he told us to do some more research, comparing toggle switch and repressilator. (http://en.wikipedia.org/wiki/Repressilator)

Also, we decided to make lists of our ideas and decide one among those by the next week.

• 03.14.2012

Another feedback arrived:

1. Pyriproxyfen, the substance that kills mosquito work as juvenile hormone analogue to prevent larvae’s development. However, we need a way to synthesize this substance other than in chemical way.

2. Toggle switch seems to be a similar system with the repressilator. It would be interesting to create microorganism with circadian rhythm or biological clock. We will discuss more about how to use it.

3. Cobalt buster seems to be unfeasible only with a few heat-resistant genes.

• 03.16.2012

We did some research about what our professor wanted us to do about toggle switch. Toggle switch is a similar system with repressilator, and the only difference is that toggle switch has two loops repressing each other and repressilator has three. And we thought circadian rhythm idea was really great.

Also, we developed our previous idea of making glowing bacteria using mechanical sensors. We thought of making a grid with 10*10 or more spaces, place gfp, and let each space have signal of 0 or 1, controlling the coloring of the bacteria in each space. It would be sort of like making the bacteria-neon sign.

Then we got reply from the professor. He told us that mechanical sensor would be found if we search previous iGEMs. For the grid idea, it would be possible with e.coli with toggle switch and turn it on or off.

• 03.17.2012

(comments from professor & advisors)

1. MIT team in 2010 iGEM made a system with mammalian cell that responds to the pressure. It is uncertain whether we can apply eukaryotic promoter to the prokaryote or not. Also, it wouldn’t be so easy to find a system in which microorganism responds to the physical stimulus. Instead, it might be possible to use ‘heat-sensing’ property, shaking it to raise the temperature and making it respond.

https://2010.igem.org/files/poster/MIT.pdf (MIT 2012 iGEM poster)

2. To develop more about the idea of coding the grid system with toggle switch, we can use the fluorescent-protein as a reporter for the switch. When switch is off, it will have value of 0 and 1 when it is on. We can use filter paper and put only on some of them the inducer that turns the switch on, and place it on e.coli. Then, it will have fluorescent-protein appearing on only some of filter papers, and when we send it, receiver can use the wave that excites the protein to get the message.

We will be meeting with the professor next Wednesday (3/21) and make final decision on our topic for the iGEM project ☺

• 03.18. 2012

Professor Choi commented that Electronic display using bacteria seems to be the best project for us to experiment.

He said that “The example of the code below is the “on only” experiment which doesn’t need toggle switch to turn it on and off.

The other possible suggestion is that first, make the circuit that works with toggle switch, and in the 96 well plate, control on-off of the each well and make the fluorescence oscillate (on and off). Then it would seem like electronic display.

I will discuss it with assistant and tell you whether it is possible or not. You can talk with him on Wednesday and make final decision.”

• 03.19.2012

We organized our brainstormed ideas before we have a meeting on Wednesday:

Sunblock utilizing bacteria

Resisting mosquitoes

Electronic display using bacteria (and toggle switch)

cobalt detection (modifying the operon in order to create a circuit that has cobalt as the inducer)

glowing bacteria with heat sensor

lactose intolerance

cholesterol degradation

glowing bacteria

plastic degradation

synthesis of flavor

methane sensing

We are also organizing wiki with the ideas that came up till now.

• 03.23.2012

First, we made decision on our research project! We are planning to work on bacteria-display.

We also uploaded wiki with what we discussed with the assistant on Wednesday.

Professor Choi commented that-

Substance that allures mosquitoes applies not only to mosquitoes but to all the insects, so it won’t work specifically to the mosquitoes.

Biosynthetic pathway isn’t well known, and the substance itself is already commercialized in the market, so it doesn’t seem to have much meaning.

Also, 1. Try making team logo 2. Upload team introduction to wiki.

• 03.25.2012

We asked several questions.

1. 96 well plate has a lot of wells each, and we are curious how gfp in separate wells uses quorum sensing to synchronize their oscillation periods.

2. You told us that the period that fluorescence appears and disappears is the same, so that we should use a video; we are curious how long it goes!

• 03.27.2012

Instructor told us that our logo seems to be simple and nice^^ and added that it would also be nice to make a logo that represents the project. He also told us that for iGEMHS, the part registration is not required; igem homepage says that special awards are given to teams who register parts with “new, cool and important function”

And here are the answers:

1. Synchronization of gfp expression in one well is explained in the paper linked below.

In Fig1(a) diagram, luxI promoter that is originally used in quorum sensing can be modified to function as repressilator.

http://www.nature.com/nature/journal/v463/n7279/full/nature08753.html

2. According to the paper, repressilator has about 150 minutes long period, and quorum sensing about 70 minutes. But it can differ according to the laboratory environment, so we will have to measure it ourselves during the experiment.

We should first search the parts related to repressilator or toggle switch, and see if we can utilize it.

• 03.28.2012

We didn’t really understand why quorum sensing has 70 minutes of period, so we asked the instructor.

In fact, reading the paper was an excruciating process but the result was worth it.

1. If we use E.coli with shorter period then we can reduce the time; does it mean that the period can be shorter than 70 minutes?

2. We found a picture for repressilator, but it says that c gene at the end represses gfp, so we aren’t really sure what’s right…

3. We are looking up parts registry site to find gene that goes into repressilator, but we are not sure what gene is needed☹ Are we using the same genes that Elowitz and Leibler used?

• 03.29.2012

Instructor sent us two papers, the one with Elowitz and Leibler & the one that is linked above.

He also told us that reducing the oscillation period shorter should be considered after we check that the system works properly. He said: “About gfp repression, you will know better if you read the paper. Based on the picture that you sent me, TetR gene is repressing the gfp expression, and gfp is expressed when λ cI gene is expressed. Also, I think that it would be too hard to create a new promoter and make oscillation system. It would be both easier and much certain to use the same method as Elowitz and Leibler’s. If you search ‘igem repressilator’ on google, you’ll see that there were many previous igem teams who used the same circuit. Laboratory schedule isn’t decided specifically. I’ll tell you as soon as I can after I discuss it with the professor.”

• 03.30.2012

We asked several questions again!

1. Both two papers are about gfp oscillation occurring in one colony; I’m curious whether we have to connect every single cells in 96 well plate into one colony

2. When doing the lab, is the trapping chamber necessary for making critical cell density as shown in 2010 paper? If we do so in 96 well plate, then how can we control flow rate?

3. In 2000 paper is the IPTG inducer that has to be inserted from the external environment? And moving the cell from place with IPTG to without IPTG resulted in the occurrence of gfp fluorescence oscillation?

On the other hand, AHL is substance synthesized by luxI, so amount of AHL also oscillated in the 2010 paper? Then I’m curious that if we have to use autoinducer similar to AHL.

4. I also want to ask if substance that is used to do cell-to-cell coupling wasn’t used in 2000 paper. We haven’t been able to read closely the previous igem projects yet. Always thank you for kind reply!

Apr 2012

• 04.02.2012

We kept reading a thesis by Elowitz & Leibler.

We were curious about why the oscillation slows down suddenly at the post-septation phase. (Then, doesn’t the oscillation happen in the E. coli after about 600 minutes, as shown in the graph?) Further, it was hard to understand why only about 40% of the cells exhibits oscillation behavior. In the thesis, it is stated that transient pulse of IPTG might be capable of synchronizing, because IPTG hinders the repression of LacI. We do not understand why. We asked these questions to professor Choi.

• 04.03.2012

This is the reply from professor Choi☺

According to the thesis, when the cell (E. coli) enters the stationary phase, the oscillation stops. (p.335 first paragraph, last sentence). The reason for only 40% of the cells showing the oscillatory characteristic may vary. Fundamentally, the oscillation is the result of complex cellular metabolism, not simple chemical reactions, so the oscillation may be highly affected by various external circumstances, such as the size of the cell, metabolism rate, diffusion rate, etc. So, as the time that the cell is exposed to IPTG becomes longer, synchronization will be hindered more, due to the same reason. I think making the circuit is not very difficult, but obtaining preferred results will not be a easy thing. We’ll make up the schedule to proceed experiments for building the circuits with your instructor.

• 04.05.2012

We thought about make oscillation of GFP expression in one colony in a 96-well-plate and size of the colony. Instructor told us that the thesis about the repressilator is about oscillation in a single E. coli cell, and the other thesis is about synchronization of oscillations in a cell colony, not a single cell. In our experiment, we will use the 96-well-plate to raise the cells in different wells as independent colonies. We will not make the whole plate as a single colony.

We were also curious about use of ‘trapping chamber’ in order to create the critical cell density. Our experiment will be conducted inside a 96-well-plate; we wonder how we could control the flow rate inside the plate, if we use the same mechanism as the ‘trapping chamber’ in order to obtain the critical cell density. We concluded that it will be quite difficult to use a trapping chamber to make a continuous culture. Instead, we will use batch culture system to raise the cells.

We asked our instructor whether we understood the theses correctly: is IPTG an inducer that must be injected from external source? And because AHL is a substance that luxI synthesizes, the amount of AHL oscillates together, right? Then, if we use quorum sensing in the experiment, do we need to use autoinducer, such as AHL?

He replied that IPTG is a chemically synthesized substance, and it is to be injected from external source. AHL is auto-synthesized. Therefore, the concentration of AHL also shows oscillation, and we do not need to put in AHL.

We also discussed that due to the difference in the period of oscillations among the cells, preferred results may not come out in our experiment. But, because repressilator parts are already registered in the parts registry by previous iGEM teams, the experiment is relatively easy to conduct right now. Instructor said that we will start preparing for the experiment and order the parts from the part registry, so we should design a theoretical model of our experiment to see if our experiment is actually plausible.

• 04.06.2012

Designing the experiment was quite a difficult process than we thought. So we asked professor for some help: 1) Is there any method to regulate the difference of oscillation period among individual cells? (Expect for putting in IPTG at the same moment)

2) If we conduct the experiment with a single cell, will the GFP expression be bright enough to be seen with naked eye?

3) Is doing the quorum sensing more difficult than building the repressilator circuit?

• 04.11.2012

We got answers to several questions that we asked the professor.

1) We can start the laboratory from the third week of April. First, we’ll make oscillation using repressilator or quorum sensing mechanism.

2) The instructor will be preparing the experiment plan, and doing the experiment approximately once a week will be appropriate.

3) If we conduct the experiment with a single cell, a fluorescence microscope is needed to observe the expression of GFP.

4) The circuits are not difficult to build, if all the parts are available. (but the circuit’s viability is a different issue)

5) The synchronization of oscillation seems to be difficult, but we will try.

6) If possible, the team members should do an in silico model experiment, using the differential equations stated in the thesis. Through the modeling, determine the parameters that the amplitude and frequency of the oscillation can be differed.

• 04.13.2012

However, due to pending AP exams that we have to take, we won’t be able to attend the lab from last week of April to the third week of May.

Also, we are going to try studying the differential equations.

• 04.25.2012

We again asked some questions on the model that we are going to use.

1. What is the reason for a repeated use of luxPL and luxPR promoters?

2. What is hybrid promoter? (It says that the promoter I751502 in front of luxI is a hybrid promoter.)

3. I wonder if the luxR-AHL complex is readily degraded. Is the degradation rate similar to that of AHL?

4. Can you explain more specifically about the protocol?

• 04.26.2012

E-mail from the instructor:

Instructor sent us e-mail with the answers to our questions:

1) luxPL and luxPR are different promoters with different characteristics. (luxPL: a constitutive promoter, lux PR: a promoter inducible by AHL-luxR complex) For luxR protein, because the protein needs to be continuously expressed, luxPL promoter is used, and for GRP, luxI, and Aiia proteins, oscillations in the protein concentration is required, so luxPR is used.

2) Hybrid promoter is not a component that we will use in our experiment. I1751502 is a combination of luxPR promoter and lac operator site; it maintains the original characteristic of luxPR (inducible by AHL-luxR complex) and has lactose-inducible characteristic at the same time.

3) Both luxR-AHL complex and free AHL degrade at a similar rate with that of Aiia. When both proteins are degraded, GFP expression turns off.

4) The standard protocol of cloning will be easier for you to understand if you read the explanation from the iGEM homepage.

• 04.29.2012

Oscillation model wasn’t an easy thing to understand completely; we had endless questions! We explained some parts that we understood, and then asked the instructor and professor what we didn’t understand yet.

As the luxI promoter becomes stronger and the amount of AHL becomes greater, the reaction rate increases (because of increased amount of luxR-AHL complex), and therefore, it is expected that the oscillation period will be shorter.

Then, 1) Is it also predictable that more GFP will be expressed as well?

2) Is it possible to predict its amount quantitatively?

In the wiki of Team Wageningen, it is stated that due to the tendency of interconnected positive and negative feedback loops to reach a steady state rather than sustaining oscillations, the external conditions need to be precisely controlled in order for the system to produce synchronized oscillations. Then, 1) How can we maintain the cell density for a long period of time?

2) Or, is it unnecessary to artificially maintain the cell density, because quorum sensing continuously occurs after certain threshold density?

• 04.30.2012

Professor e-mailed us again and told us that 3A assembly kit will be arriving this week, so that we can use it in the experiment (http://partsregistry.org/Help/3A_Assembly_Kit) : Also, the calendar of events can be looked up in the link shown, and we had to keep track of schedule that we have to go through. ( https://2012hs.igem.org/Calendar_of_Events) And the judging criteria can be found here ( https://2012hs.igem.org/Judging).

We now need to decide the time for our experiment and start working on it. Instructor will help us throughout the experiment, and he will be including the experiment for observing the growth curve of E Coli and measuring/observing fluorescent proteins.

May 2012

• 05.05.2012

We learned about biobrick standard assembly and 3 antibiotic (3A) assembly. Standard assembly is a two way ligation with two biobricks and a vector. 3A ligation is a three way ligation using three biobricks, vector, and three antibiotics to get only the successfully transformed cell. When each biobrick part is cut with different enzymes, they possess sticky ends that enable them to combine. However, once they are combined, they make mix-ends which would not be recognized by any of the restriction enzymes.

http://partsregistry.org/Assembly:Standard_assembly

We went over biobrick standard assembly / 3A assembly

3A assembly relies on three way ligation for assembly rather than two way ligation between a part and a part + vector molecule(standard assembly) different sticky ends are able together even if they are cut with different enzymes but such part become mixed-ends, which means they would not be recognized by any of the restriction enzymes.

• 05.10.2012

We further studied assembly methods in the website below:

http://www.bio.davidson.edu/courses/molbio/BioBricks/BioBricks.html

According to this website, all restriction sites are palindromes, top strand 5’ to 3’ being identical to the bottom strand 5’ to 3’, which is called sticky ends. Mixed sites are not palindrome, so they cannot be cut by any restriction enzyme.

• 05.12.2012

We learned about what is a linearized plasmid backbone. By using linearized plasmid backbone, the remnant cut with EcoRI and PstI is short that it will not ligate. And this allows to build high quality construction plasmid backbone without purifying the cut fragments remaining after PCR.

2012 DNA Distribution came with a set of linearized plasmid backbones: pSB1A3, pSB1C3, pSB1K3.m1, and pSB1T3.

• 05.21.2012

We further studied about protein purification using gel electrophoresis in standard assembly. Protein purification is series of processes intended to isolate a single type of protein from a complex mixture. pH graded gel / ion exchange column / size exclusion chromatography / SDS-PAGE are examples.

• 05.22.2012

We read an article about the protocol from iGEM 2011 Team Wageningen wiki, and wondered if there is any process from the protocol of Team Wageningen that we are not planning to use.

Also, we could find many kinds of protocols, other than the one that Team Wageningen has used, such as:

· Preparing chemically competent cells · Preparing TSS buffer · Transforming chemically competent cells · Preparing chemically competent cells (Inoue) · Transforming chemically competent cells (Inoue) · TOP10 chemically competent cells

So we asked the professor what we are planning to use among them.

Also, there are some questions about the article written by Team Wageningen:

1) What does OD stands for?

In Wikipedia, it is indicated that OD stands for either ‘optical depth’ or ‘optical density’, but we cannot figure out in what meaning was ‘OD’ used in the protocol.

2) What does ‘OD 600nm of 0.3’ mean?

3) And, why should the OD value changed into 1.0~1.5 later on. What influence does OD have in the experiment?

• 05.23.2012

And our instructor gave us the answers for our questions☺

He told us that OD stands for ‘optical density’. The OD value is proportional to bacterial cell number. The cell number is larger if the OD value is larger.

From the OD value, we can predict not only the number but also cell condition. During the phase in which cells grow actively, the OD value increases continuously, but after certain period of time, the OD value does not increase any more.

In this phase, the cells stop cell division and get themselves prepared for death.

When we conduct the experiment, it is better for us to do it with healthy, young cells, rather than old, weak cells.

Therefore, in the protocol, the OD number is controlled to regulate the cell number or to use cells in good conditions.

Also, he asked us when we could visit the lab. And if we have time, he is going to tell us the experiment protocol starting from making chemically competent cells. He told us that he is currently working on the cloning of the cells.

• 05.30.2012

Our team member Ju Yeon Han and our instructor Hong Jae Park ( participated in the iGEM online meeting at Second Life. During the meeting, we could talk to the iGEM headquarter and another iGEM high school team from Mexico. At first, we talked about what we experienced through iGEM; our team member said that she learned a lot from reading scientific papers and learning more about synthetic biology that we can barely learn from school. The participants of the online meeting also exchanged ideas about socializing at the jamboree; some ideas were suggested, such as running a café, Xbox game room or a basketball court.

Then, Ju Yeon asked a question about organizing the wiki; igemHQ told her a way to create new pages in the wiki. Our instructor also asked a question; it was about a problem in the assembly process. Our instructor tried to assemble the parts using the 3A assembly method, but unexpected results kept coming out; for example, red colonies were found, although white colonies were expected to be made. Jimmybly (the Mexican team) was also having some problem regarding the linearization of plasmid and was having some unexpected results, such as finding white colonies when red colonies are expected to be present. Also, our instructor said that when he cut luxR, luxI, aiia and GFP from psb1A2, the vector size of luxR turns out to be unexpectedly large; he later on found out that the part that we were using in our experiment was actually inconsistent.

In the later part of the meeting, the participants of the meeting exchanged ideas on the issue about the ‘motivation’ of participating in iGEM and some advices to improve iGEM administration.

June 2012

• 06.09.2012

We were having hard time with cloning RBS and attaching it because its sequence was too short. But in second life, iGEM gave us a hint! They told us it would be easier if we used PCR. So we used that method. However, we still didn’t know the perfect temperature of PCR, so we made several samples and cloned it in various temperatures starting from 63 celcius to 68 celcius.

• 06.16.2012

We decided to use two vectors: One having Lux PR, aiia, GFP, and Lux I and the other vector having Lux PL and Lux R. We used vectors that are resistant to ampicillin and kanamycin, so only those cells that were successfully transformed with both vectors will survive. Also, when the biobricks are inserted between the vector, it will cut the RFP gene so the colony will be white when cultured. Otherwise, if the vector does not contain the biobrick parts that we wanted, the colony will be red.

We cloned the biobrick parts and transformed into the cell. It was very interesting. Our instructor brought us to upstairs where competent cells were stored. We learned about competent cells in our textbook but it was our first time to actually look at it. The cells were stored in -80 celcius. It was quite surprising. When we took them out, the instructor told us that the cells have to be kept in ice. Also, when transforming, our instructor told us not to speak because the cells can be infected.