Team:BIOSINT Mexico/Project
Coliroid
Background
Overview
Color Coliroid
Assembly
For the assembly, we are working with two different constructs, both in pSB1C3, with differentiated functions. The plasmids are show below.
Figure N° 1. pSB1C3 with construct for constitutive expression for PCB (Ho1 and pcyA), and regulated expression of spisPink attached upstream to FixK2 promoter.
Figure N° 2. pSB1C3 with construct for constitutive expression of red and blue light photoreceptors (cph8 and YF1), and regulated expression of amilGFP attached upstream to FixK2 promoter.
Devices
Several devices were designed and built, as shown in the next image.
Figure N° 5. Color-Coliroid. The figure above show how the system works.
Photoreceptors
YF1 and Cph8 are two different photoreceptors, chimeric proteins These Are That Have the Ability to phosphorylate or dephosphorylate Their respective transcription factors. For Their constant expression inside the cell.
For this, first the coding sequences joined a sequence RBS (BBa_J61100), and then were joined together, in addition to which was added a terminator (BBa_B0030) at the 3 'end and a constitutive promoter (BBa_J23101) upstream, in the 5 '
Transcription factors
YF1 and Cph8 are responsible for regulating the activity of two different promoters, by phosphorylation of two transcription factors. Thus, a device capable of producing FixJ transcription factor associated with YF1 and FixK2 was designed. Also, ompR is a transcription factor associated with cph8, however, since endogenous ompR use, the device was assembled to produce PCB (HO1 and pcyA), which helps the assembly of this transcription factor.
To this they are joined HO1 (BBa_I15008) pcyA (BBa_I15009) and FixJ (BBa_K592005) with RBS (BBa_J61100) upstream. All these parts are joined together with a strong constitutive promoter (BBa_J23101) and a terminator at the end (BBa_B0030).
Chromoproteins
Chromoproteins Both were linked to a repressible promoter, that is associated with transcription factors, to produce light when there is.
spisPink optimized for E. coli K12 (BBa_K1684000) are joined together to FixK2 promoter (BBa_K592006), an RBS (BBa_J61100), and a terminator (BBa_B0030)
amilGFP optimized for E. coli K12 (BBa_K1684001) and joined together to FixK2 promoter (BBa_K592006), an RBS (BBa_J61100), and a terminator (BBa_B0030)
Methods
Light Cannon
A device which meets the needs of the project was designed.
This device consists of a box in which a system for temperature control is implemented also a mechanism in which not to do many things manually is implemented; only you need to move a knob to the figure you want to project in the Petri dish is changed.
The mechanism is commonly known as Geneva mechanism, it is a mechanism steps. The original mechanism consists of a rotary movement in which every time the circular piece rotates one revolution the other piece is moved one step. You can design the mechanism so that it has three or more steps:
The Geneva mechanism that was designed consists of four steps in which each step an image that will not let light pass through it will. In each quarter revolution will move one step design that was given. A knob to make the move from outside the box, if you move the knob one full turn will become the first step and therefore the first figure is attached. This knob is manually moved to allow time for the bacteria to receive wavelengths.
Care in designing the components of the pieces do not collide with each other to perform the rotary movement had:
The box consists of a drawer that he can change the petri dish:
Top box has a hole through which a projector is attached to occur with an RGB combination the desired wavelength:
The projector, the top hole of the box and figure to be projected are aligned.
The material of the box must be insulated, because you do not want to have a heat exchange with the surroundings.
Have a resistance on the back of the box, it is desired to increase the temperature inside the box. Between the top where the Geneva mechanism and the laboratory dish is separated from the back in case the resistance emits unwanted wavelengths becomes hot is located. It should be a material with high thermal conductivity between the two sections to be rapidly spread.
The sensor would be at the top of the box.
The electronic system consists of a temperature sensor is one that would be used as predesigned. Obtained from the LM335 datasheet from Texas Instruments:
The circuit must be outside the box by security components except heat resistance and the temperature sensor.
Results
spisPink & amilGFP optimized to Escherichia coli K12
After completing the insertion of gBlocks (IDT) designed (spisPink and amilGFP), into the plasmid pSB1C3, by following the relevant protocols; competent cells were successfully transformed and consequently the removal of plasmids (spisPink into pSB1C3) BBa_K1684000 and (amilGFP into pSB1C3) BBa_K1684001 was performed.
The Figure 6 presents a 1% agarose gel which contains two samples, by triplicate, next to the ladder. In line 1, 2 and 3 are located the triplicate samples of spisPink piece, optimized for E. coli K12 (BBa_K1684000), with a size of 2789 bp. Likewise on the line 4,5 and 6 are repeated samples of the piece amilGFP optimized for E. coli K12 (BBa_K1684001). For both BioBricks, the electrophoresis results correspond to the theoretical size, greater than 2500 bp and less than 3000 bp; also it can be seen that there is no variation between samples, replicas, indicating the reliability of E. coli K12 transformations.
After completing the insertion of gBlocks (IDT) designed (spisPink and amilGFP), into the plasmid pSB1C3, by following the relevant protocols; competent cells were successfully transformed and consequently the removal of plasmids (spisPink into pSB1C3) BBa_K1684000 and (amilGFP into pSB1C3) BBa_K1684001 was performed.
The Figure 6 presents a 1% agarose gel which contains two samples, by triplicate, next to the ladder. In line 1, 2 and 3 are located the triplicate samples of spisPink piece, optimized for E. coli K12 (BBa_K1684000), with a size of 2789 bp. Likewise on the line 4,5 and 6 are repeated samples of the piece amilGFP optimized for E. coli K12 (BBa_K1684001). For both BioBricks, the electrophoresis results correspond to the theoretical size, greater than 2500 bp and less than 3000 bp; also it can be seen that there is no variation between samples, replicas, indicating the reliability of E. coli K12 transformations.
Figure 6: Agarose gel with spisPink and amigGFP samples, optimized for E. coli K12 (x3)
RBS and Optimized biobricks
In Figure 7 different samples of parts used for transformation of competent cells; these two parts are composite biobricks, the first is composed by RBS (BBa_B0034) and the optimized chromoprotein sequence spisPink for E. coli K12 (BBa_K1684000), while the second is composed by a RBS (BBa_B0034) and the optimized chromoprotein sequence amilGFP (BBa_K1684001 ).
The agarose gel shown in Line 1, the negative control; Line 2, belongs to the purified DNA of pGLO (5371 bp), was run as a positive control. Line 3 and Line 4 contain duplicate products of BBa_K1684002 (BBa_B0034 + BBa_K1684000) biobrick, both results match the size of the plasmids, 2800 bp. Line 5 and Line 6 contain duplicate BBa_K1684003 (BBa_B0034 + BBa_K1684001) biobrick; however, a positive result is observed only in Line 5, with size of 2822 bp, while in line 6 only presents contamination, without the presence of any genetic material.
Figure 7: Agarose gel with BBa_K1684002 (x2) and BBa_K1684003(x2) samples, positive and negative controls
Optimized biobricks with Terminator
Finally, in Figure 8 samples composite parts are presented, the first BBa_K1684004, is composed of the chromoprotein sequence optimized for E. coli K12 spisPink (BBa_K1684000) and a terminator (BBa_B0010); the second part is composed of the chromoprotein optimized sequence amilGFP (BBa_K1684001) and the same terminator (BBa_B0010). This agarose gel shown on Line 1 and Line 2, the biobrick BBa_K1684004, in the first case the band run corresponding to its size, 2869 bp; however, on Line 2 the result was not the expected and only contamination was obtained.
In Line 3 and 4 results, was run the composite BioBrick BBa_K1684005, in the first case the result was not successful; In Line 4 however, the band is present with the respective size, 2890 bp, as a positive result. Finally on Line 5 is presented only the negative control.
Figure 8: Agarose gel with BBa_K1684004 (x2) and BBa_K1684005(x2) samples, and negative control.