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Team:ITB Indonesia/nb-wetlab
From 2014.igem.org
June
Week 1
In the first week of June we observed the laboratory. We made a list of materials that we need for future wetlab activities. We prepared LB medium, searched the antibiotic optimization method and made competent cells using CCMB 80 buffer. We also sterilized all of the equipment needed. We already decided our idea that is engineer bacteria that can degrade plastic. Previously, we had four main ideas:
1. Microalgae biofuel
2. Synthetic bacteria that can degrade plastic
3. Urine bio-sensor
4. Cyanide bio-sensor
After competent cells were ready and antibiotic optimization had done then we had plan to try re-hydrated some parts and transform it to our Escherichia coli DH5α competent cells. We also had decided our construct so we would ready to order some synthetic parts and ordered it to iGEM HQ especially for some parts that not provided in the 2014 parts.
Week 2
This was our first time to see the iGEM distribution kit plate which was so exciting. We would make a trial to transform iGEM 2013 distribution kit using our competent cells. That transformation was not good on first, second, and third trial. Then we concluded that we must try the iGEM distribution kit plate 2014. We also tried to test our competent cells efficiency and we did not face any problem with our competent cells.
Week 3
Finally, we re-hydrated parts from iGEM kit plate 2014 which had many copies to make sure our method was optimal enough to transform those iGEM parts. We had already a plan to make four systems for our bacteria to degrade plastic. We divided our team into four groups. Each groups focused to each systems. We tried to transform BBa_K592025, BBa_B0015, BBa_B0034, BBa_I0040. The transformation did not have any validate colonies.
Week 4
We targeted to transform parts from iGEM distribution kit. Finally, we could see our red colony from RFP parts. So, we made liquid culture and wait until 16 hours. Then the medium turned into red. We tried to isolate the plasmid using Alkaline Lysis Method. The confirmation of the plasmids isolated were by electrophoresis, restriction and PCR method. The result was positive.
July
Week 1
Finnaly our constructs are already fixed. We ordered our synthetic gene for our main construct and ordered some parts from iGEM Headquarters. Our competent cell was also ready for transforming the other parts that we will use for our construct. We rehydrated BBa_K592025, BBa_J04450, BBa_C0040, BBa_B0017 and BBa_B0015, the colonies was growing well and ready for plasmid isolation. All of the plasmid was confirmed using restriction method and the result was positive. In this first week of July we learnt more about 3A assembly since we are ready to make our construct as well.
Week 2
We are ready to construct our building blocks. But first, we made some optimization method for double digest restriction reaction including for time incubation and reaction compotition. We also received our parts that we had ordered from iGEM headquarter BBa_K103006, BBa_K228004, BBa_K936024, BBa_K339010. We also tried to build our construct using two methods: standard assembly and 3A assembly. Still not work well. In this week we also elaborated and had brainstorming for our next method after our construct had been done.
Week 3
We were ready to try 3A assembly again, we made four different ligation composition to make sure which one will work well. After transformation, finally we got few colony in our petri disc, ready for plasmid isolation. After confirmation for all of the plasmid, we got no right colony yet. We then tried again using PSB1C3 with Rfp for the plasmid backbone instead of linearized plasmid to make it easier for colony selection using red-white screening. Still not got our right construct. Let the challenge begin and let’s do the best for it.
Week 4
Challenge accepted, we did our 3A again. We discussed about our failure for the 3A this two weeks and got some conclusion. Finally for this trial we got some colony which was the right construct BBa_K1387002. We then found the optimum ligation composition and reaction. Ready for another construct for reporter and converter module while waiting for synthetic gene for our main construct.
August
Week 1
Luckily, the primers were arrived. We ordered universal and spesific primer to check our construct using PCR technique. For optimization, we used gradient PCR technique. By using this technique, we were able to get a good results. Unfortunately, many of our construct were still in pSB1K3 backbone, so we must translocate it to pSB1C3 backbone.
Week 2
In this week we are trying to make our 3A assembly again since we still have some gene construct that must be finished before our synthetic gene comes. From the 3A assembly we got several colony that ready for plasmid isolation. After the confirmation through PCR methode, we did not get the right colony yet, so we must try the other compotition in ligation compotition, because we did not face any problem in transformation. After trying any ligation reaction , we got the optimal compotition for some construct, but the other still become our big mystery. In this week, we are also want to try the construct from UC Davis for Lc Cutinase BBa_K936020 and ethylene glycol converter module BBa_K936024. We did plasmid isolation from that part and transform it to the E.coli BL21 (DE3) We try to test the gene expression and test the product through SDS-PAGE method and we can see the protein band from both of the construct but not as the thick band. We also try to sequencing the positive construct in this week.
Week 3
Our target in this third week of august is to finish all of the construct except the construct that need synthetic gene due the parts submission deadline is coming. Restriction, purification gell (if needed), ligation, transformation, plasmid isolation, and confirmation is become our routine activities. In this week, we also learn more about how to express our protein, how to test our construct, how to measure the construct activities and did SDS-PAGE analysis.
Week 4
We focused to make a simple trial for our method to test the protein band of cutinase, and conversion methode, and discuss about our construct characterization especially for OmpA-LcCutinase construct (PET degradation module) as our main construct. We also discuss for synthetic gene cloning method. In this week we also focused on finishing our construct in PSB1C3 backbone and ready for shipment. Brainstoarming also become our routine activities because we found some failure in this week and feel not ready yet facing the parts submision deadline. We also try to elaborate how to make a modeling for our system and discuss with the expert.
September
Week 1
We have to prepare our parts submission form and check all of positive construct to make sure the condition of the construct is still good. In this week, synthetic gene, the thing that we waiting for one and half month finnaly arrived in our campuss. We try to make optimal reaction for our limited stock of synthetic gene. We try to clone our synthetic gene into some backbone including some of commercial clone vector and PSB1C3 of course. No collonies observed, so desperate and we discuss and evaluate our method and take a note of some mistake that we did in this method. Fortunatelly we have primer for our synthetic gene so we could do amplification using PCR for our synthetic gene. We also make new competent cell and trying electhrophoration transformation method instead of common heat shock methode. Still, no colonies observed.
Week 2
We try to clone our synthetic again. We must be succes in this week to step forward finishing our main construct. We try several method and try to use overnight ligation method. Finnaly, we got the positive colony, so happy. We also checked our plasmid again and found that some of our construct disappear but the other is still in good condition. We also make a optimization for PCR using touchdown and temperature gradient method to check the good temperature condition for the primer.
Week 3
We did PCR for construct confirmation for the collonies through crude PCR method using the optimal annealing temperature. We also did plasmid isolation for the positive colonies. We also make the method plan for PET degradation characterization using our construct and compared to UC Davis construct. We make some method for measurement including pH detection, ethylene glycol detection using dichromic acid method, and check the correlation between cell number in PET degradation rate.
Week 4
We submitted some of our parts including BBa_K1387000, BBa_K1387002 and BBa_K1387006 In the 11 days remaining for parts submission, we still try to build our construct and start to think the future development of our system. We also try to help Brawijaya team to cloning some of their parts and synthetic gene. We will start our parts characterization using several method including ethylene glycol assay using chromatic acid, pNPP assay to check the activity, growth curve, SEM, and qualitative assay using tributirin agar method.
October
Week 1
The sending parts week for second term. three parts is ready to send including BBa_K1387001, BBa_K1387006 and BBa_K1387005. Our dry lab team is also ready for modelling. We got some parameter to make the modelling. We also got some idea for our future system.
