Team:OUC-China/Improve
Improve
Overview
This year, we have created a brand new family called Csy4 family on the basis of an existing part BBa_K1062004. We redesign four Csy4 mutants by point mutation to form this family, whose capabilities of cleavage and recognition are different from each other. As an important role in our project miniToe family, we have tested them by several ways. We have proved that our system can work well by using Csy4 family. Now Csy4 family is an improvement and has been shown to work well in our system.
Proof of functions about Csy4 family
We have done three kinds of experiments to help us confirm the function of the Csy4 family. Our aim is to get some new Csy4 mutants with different cleavage capacity, so we specifically tested this aspect of them. For testing our system, we use sfGFP as our target gene. Our expectation is that the fluorescence intensities of sfGFP can vary upon the rates of Csy4s’ cleavage. That means we have improved four new parts which present various expression of target genes.
Prediction
Before the experiments, we have proved our ideas by model. The predication below shows the possibilities of different expression levels by different Csy4 mutants. So the model help us to get more information for our improvement deeply this year!
Fig.2-3 The predication: The fluorescence intensities by different Csy4 mutants along with time
The result of miniToe Motility detection system
Proof of functions
As we shown before in the first system, we have created a new method to regulate to downstream gene expression named miniToe. And we also have proved that our system can be enlarged. So we have created miniToe family in the second system.We believe that miniToe is also a good tool which can be apply to study of molecular mechanism. Scientists may puzzled with the functions of certain gene or protein when first discover it. Now one common method to study single gene is knock out or knock in. In this way, organisms without one gene show the lack of forms or functions. But if we want to know better about the gene functions, we may need different level of the gene expressions.
By using our system, the motility of E.coli can be regulated. As we know, MotA provides a channel for the proton gradient required for generation of torque. ΔmotA strains can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.
So we have done some works to test our system in dealing with the real-world problems by E.coli motility detection experiments. We construct our circuit by putting the motA behind our miniToe structure. So the motA as a target gene can be regulated by miniToe.
Five groups have been set, three test group and two control group. And the result shown below have proved that our system can work as expectation.
Fig.4-1 The control groups including positive group and negative group. Plates were inoculated with E.coli RP437 (A1, A2, A3) which have motility and they move everywhere in the plates. The plates on right are ΔmotA (the motA-deletion strain) (B1, B2, B3), E.coli RP6666 which have no motility so the strains stay on the center. We have three biological replicates in the experiment.
Fig.4-2 The test group-1. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe structure).Without the gene motA, the E.coli cannot move. And the Csy4 have no big influence on strain compared with the strain, ΔmotA. We have three biological replicates in the experiment.
Fig.4-3 The test group-2. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe structure but no Csy4). The circuit is on the control of miniToe and it down stream motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.
Fig.4-4 The test group.
The result of miniToe Motility detection system
Proof of functions
As we shown before in the first system, we have created a new method to regulate to downstream gene expression named miniToe. And we also have proved that our system can be enlarged. So we have created miniToe family in the second system.We believe that miniToe is also a good tool which can be apply to study of molecular mechanism. Scientists may puzzled with the functions of certain gene or protein when first discover it. Now one common method to study single gene is knock out or knock in. In this way, organisms without one gene show the lack of forms or functions. But if we want to know better about the gene functions, we may need different level of the gene expressions.
By using our system, the motility of E.coli can be regulated. As we know, MotA provides a channel for the proton gradient required for generation of torque. ΔmotA strains can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.
So we have done some works to test our system in dealing with the real-world problems by E.coli motility detection experiments. We construct our circuit by putting the motA behind our miniToe structure. So the motA as a target gene can be regulated by miniToe.
Five groups have been set, three test group and two control group. And the result shown below have proved that our system can work as expectation.
Fig.4-1 The control groups including positive group and negative group. Plates were inoculated with E.coli RP437 (A1, A2, A3) which have motility and they move everywhere in the plates. The plates on right are ΔmotA (the motA-deletion strain) (B1, B2, B3), E.coli RP6666 which have no motility so the strains stay on the center. We have three biological replicates in the experiment.
Fig.4-2 The test group-1. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe structure).Without the gene motA, the E.coli cannot move. And the Csy4 have no big influence on strain compared with the strain, ΔmotA. We have three biological replicates in the experiment.
Fig.4-3 The test group-2. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe structure but no Csy4). The circuit is on the control of miniToe and it down stream motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.
Fig.4-4 The test group.