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Part:BBa_K081014:Experience
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Part BBa_K572001 - RFP Generator with Different promoters regulated by MerR, sensible to Mercury
Our team, UFAM-UREA_Brazil, used this BioBrick to measure new promoters regulated by MerR efficiency! It works pretty good! Thank you Lorenzo Pasotti and Paolo Magni!Check out the results!
Our team (UFAM-UEA_Brazil) worked improving the Mer operon expression to increase bioremediation in E. coli through novel mer promoters sequences. For it, we primarily characterized the MerR expression under control of different promoters from Anderson Collection (BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033) through the repression of RFP (BBa_K081014) production, in a synthetic genetic circuit represented bellow.
On the first experiment, we used the novel regulated promoter designed by our team BBa_K2123109 (Stationary growth phase promoter with downstream mer operator) to measure the RFP repression by MerR under control of BBa_J23100, BBa_J23104, BBa_J23106 e BBa_I142033 constitutive promoters in solid LB media, as you can see below.
You can analyse by the RFP expression and thus fluorescence intensity that the samples greater repressed by MerR was the ones under control of BBa_J23100 and BBa_I14033 constituve promoters. So, we measured the RFP expression using Chamaleon Spectrofluorometer with and without MerR repressor protein under control of these two constituve promoter. The results are presented in the graph 1 below.
Graph 1: RFP expression with and without MerR regulator under control of BBa_J23100 and BBa_I14033. Control bacteria is DH5-alpha without any plasmid vector.
As we can see, between BBa_J23100 and BBa_I14033, the best repressed was with BBa_J23100 constitutive promoter. So, we selected BBa_J23100 to controls MerR expression in our synthetic genetic circuits. We made also other experiments to understand the interactions between MerR and new regulated promoters designed by our team, aiming to reach a well repressor mechanism, increasing the natural MerR regulation. In this way, we measured MerR regulation with two more regulated promoters: BBa_K2123102 and BBa_K2123101 - Tac promoter + overlapped and between mer operator, respectively expression RFP. The MerR regulation mechanism was characterized by repressing RFP expression and therefore reducing fluorescence intensity according to the regulator efficiency. We measured it also utilizing Chamaleon Spectrofluorometer. The results are presented in the graph 2 and 3 below.
Graph 02 and 03: RFP expression with and without MerR regulator under control of BBa_J23100 constitutive promoter, repressing BBa_K2123101 and BBa_K2123102 new regulated promoters. We achieved almost a totally repression with this BioBrick part! Awesome results, as you can see in the figures below.
Figure 04: RFP expression regulation by MerR. </html>
Part BBa_K572001 - RFP Generator with PcstA Carbon Stress Promoter
Applications of BBa_K081014
Experiment 1 - performed by Lorenzo Pasotti and Mattia Quattrocelli
This generator was contained into pSB1AK3. The constitutive promoter BBa_J23100 was assembled upstream of the generator using BioBrick Standard Assembly.
After ligation reaction we transformed 1 ul of ligation product into 50 ul of Invitrogen TOP10 and plated transformed bacteria on LB agar plates with Ampicilline.
15 hours after plate incubation at 37°C, we put the plate on the transluminator. Positive transformants glowed under UV rays (see figure).
We also picked up some colonies from the plate using a tip and infected 1 ml of LB + Amp. After 3 hours of incubation (37°C, 220 rpm) we watched 50 ul of bacteria with TRITC channel. Results are reported in the following figure.
We can see that our RFP generator actually works.
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