Team:QHFZ-China/Parts
Overview
1. Basic part
BBa_K3007000 hucO
BBa_K3007001 PhucR
BBa_K3007005 RinA_p80α
BBa_K3007008 PrinA_p80α
BBa_K3007023 CP6 promoter
BBa_K3007024 HucR & YgfU
BBa_K3007011 URAT1
BBa_K3007012 KRAB-HucR
BBa_K3007013 hucO8
BBa_K3007014 smUOX
2. Composite part
BBa_K3007022 PhucR-dsRed BBa_K3007025 Pcp6-HucR & YgfU BBa_K3007029 PhucR-dsRed-Pcp6-HucR & YgfU BBa_K3007030 PhucR-RinA_p80α BBa_K3007031 PhucR-RinA_p80α-Pcp6-HucR & YgfU BBa_K3007010 PrinA_p80α-B0030-sfGFP BBa_K3007034 PrinA_p80α-B0030-dsRed BBa-K3007011-14 are used to degrade uric acid in HeLa cells. BBa_K300729 was used to detect uric acid (UA) in E. coli. BBa_K300731 with either BBa_K3007010 or BBa_K3007034 was slao used to detect UA in E. coli. However an amplifier module was added.
Medals
Characterization (Bronze) BBa_B0030 RBS B0030 BBa_I746916 sfGFP BBa_K2197303 hucO
Validated Part (Silver) BBa_K3007001 PhucR BBa_K3007005 RinA_p80α BBa_K3007010 PrinA_p80α-B0030-sfGFP
Improve a previous part (Gold)
Previous: BBa_K2197300
New: BBa_K3007029
After we got our composite part BBa_K3007029 that can respond to UA, we investigated whether the part worked better than a pre-existing part, BBa_K2197300. As the author team did not measured the part and declared the part they submitted could have something wrong, we decided to test the key difference of the two parts. Via analyzing, we found the HucR expression part is similar, while the most improvement of our part is using PhucR (BBa_K3007001) to control the expression of reporter gene and sense UA. Notably, considering that lac promoter is one of the most used promoter, we insert the hucO, which is the HucR binding sequence, between -35 and -10 regions of a modified lac promoter. However, in BBa_K2197300, the promoter BBa_J23106 and hucO sequence BBa_K2197303 connected end to end. As a result, we decided to construct a vector based on our vector by changing PhucR into BBa_J23106+ BBa_K2197303, to simulate the function of BBa_K2197300. As the 3’ end of lac promoter contains a RBS BBa_B0029 sequence, we retained the RBS sequence so that no more RBS is needed (Fig.1).
Fig. 1 An introduction of the strategy to compare the two parts. (A) A schematic diagram showing the main difference of the two parts. The sequence in red frame was replaced by BBa_J23106+ BBa_K2197303 to simulate the function of BBa_K2197300. (B) The sequencing data showing we had successfully constructed the vector to test the function of BBa_K2197300.
We chose three correct clones to do the experiment. We tested the response of the two parts to UA at the same time. The part that simulated the function of BBa_K2197300 could not respond to UA, while our BBa_K3007029 gave gradually enhanced fluorescence with the rise of UA concentration (Fig. 2).
Fig. 2 Responding curve about the fluorescence / OD600 to different UA concentration of the two parts. For every part, three clones was tested. Data were shown as mean ± SD. N = 3 technical repetitions.
Then we did more measurement and improvement of our BBa_K3007029. The detailed information has been shown in Demonstrate section.
