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Team:OUC-China/Result/fv
From 2011.igem.org
As you can see in the project introduction, Wuxing theory and our five QS based devices are composed with five complicated devices with similar structures. After deep deliberation and self-time evaluation, complete construction of BioWuxing with this summer is impractical, so we decided to do three-elements promoting cycle using previous biobricks and running fluorescent test as examination, if this works then other similar devices will be easily built or perhaps will be built automatically in the near future. Simultaneously, we asked for PCR-Source rhizobium to standardize Wuxingbricks and complementary LeuB gene.
First step (July)
Preparing for 2011 distribution transform and running quality examination.We transform 14 biobricks from 2011 distribution at first, after designing our circuits we request another 8 parts for device construction. You can see part details here, I will call them in specific order for short.
Second step (August)
Communication system construction before plug in standardized LeuB.
Device 1 5-4-6-2-3
Device 2 18-3-11-16
Device 3 14-19-13
During the construction, we at first use the standard method of assembling, 6-2, 14-19 assembling succeed in this way but it is quite inconvenient because of gel extraction and low transform rate. Other ligation is achieved by 3A.
Since LeuB had not be plug into the device, we made two programs of ligation, one for direct fluorescent protein test, the other for connection of LeuB. Here are the two designed programs of devices 2:
Fluorescent protein examination
Expected result: We use 5-4 as a lasI generator to produce AHLs, after adding IPTG, device 1 is on work, the molecular transferring should be activated, then the other two devices activated, reporter gene express, 3 fluorescence could be observed.
Please follow details about the examination
Since part 16 have quality problems, device 2 may not work as expected, verification of device 3 will also be seriously affected. At this time, LeuB is prepared, so we decided to go on with our work, combine communication with survival function.
After the examination, it met our expect that device 2、3 could not show supposed fluorescence, only device 1 is working showing fluorescence as expected.
Third step (September)
PCR of LeuB from BL21
PCR of SinI SinR SinIpro; RhiI, RhiR, RhiABCpro; RaiR RaiI RaiIpro
Standardization of SinI SinR SinIpro; RhiI, RhiR, RhiABCpro; RaiR RaiI RaiIpro
Building combination of communicational and survival mechanism:
*Device 1
*Device 2 The origin faulty part16 is being re-ligated from B0030 C0076 B0034 E0020,as shown below
*Device 3
Fourth step (October)
Ligate S01010 afresh by B0034 C0076 B0034 E0020 for device 2
Ligate J13002 and 6-leuB: transform the leuB generator into HB101 for leuB function test
Construction of newer three devices is almost ready.
Test the following function: signal emitting and receiving, co-survival or die together:
Wuxing’s Home (Quincuncial piles----our unique culture apparatus)
The design of this device aims to enable different bacterial strains to rely on the spread of signal molecules to communicate, by usage of common bacteria liquid medium and the filtering membrane. We designed to use an electric cutter over the plastic dish to cut five 1cm-diameter holes and insert a 6.5mm-diameter plug-in tube into each hole through filtering membrane.
Cover an upside-down round-bottomed glass dish in the former plastic dish. After sterilization, add LB medium to the dish in clean bench so that it can penetrate into the submerged membrane and flow to plug-in tubes. Then, inoculate with bacteria to plug-in tubes.
Place electromagnetic stirrers into petri dishes. Stir with the constant low speed stirring incubator.
In our device, different strains can only grow in their own culture tube, while signal molecules can transfer through the membrane and diffuse into the bottom of the dish to affect other strains.
