Description

At the beginning of the InterLab study we completed three distinct calibration protocols. At first, we performed the LUDOX Protocol in order to obtain a conversion factor to transform absorbance (Abs600) from the plate reader into a comparable OD600 measurement as would be obtained with a spectrophotometer. Next, we completed the Microsphere Protocol as it allows a standard curve of particle concentration which is used to convert Abs600 measurements to an estimated number of cells. Finally, by completing the Fluorescein Protocol we generated a standard fluorescence curve which is used to compare fluorescence output of different test devices. Completion of the calibrations ensured that we take cell measurements under the same conditions. It is worth mentioning that prior calibration, we prepared competent E. coli DH5-alpha cells and transformed them according to the standard transformation protocol. During all of the experiments we tested 8 plasmids: 2 controls and 6 test devices (Table 1).

Table 1. Parts received and tested during iGEM’s fifth InterLab Study

Table 1.

RESULTS AND DISCUSSION

1. MEASUREMENT OF LUDOX CL-X OD600 REFERENCE POINT

Using LUDOX CL-X as a single point reference allowed us to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. This is crucial to ensure that plate reader measurements are not volume dependent. After this calibration part we obtained a radiometric conversion factor (Table 2) which will be used in further Interlab study measurements.

The framework also includes a possibility of adding a selection system that reduces the usage of antibiotics (only 1 antibiotic for up to 5 different plasmids!) and an active partitioning system to make sure that low copy number plasmid groups are not lost during the division.

Applications

Everyday lab work

A multi-plasmid system that is easy to assemble and control. With our framework the need to limit your research to a particular plasmid copy number just because there are not enough right replicons to choose from, is eliminated. With SynORI you can easily create a vector with a desired copy number that suits your needs.

Biological computing

The ability to choose a wide range of copy number options and their control types will make the synthetic biology engineering much more flexible and predictable. Introduction of plasmid copy number regulation is equivalent to adding a global parameter to a computer system. It enables the coordination of multiple gene group expression.

Smart assembly of large protein complexes

The co-expression of multi-subunit complexes using different replicons brings incoherency to an already chaotic cell system. This can be avoided by using SynORI, as in this framework every plasmid group uses the same type of control, and in addition can act in a group-specific manner.

Metabolic engineering

A big challenge for heterologous expression of multiple gene pathways is to accurately adjust the levels of each enzyme to achieve optimal production efficiency. Precise promoter tuning in transcriptional control and synthetic ribosome binding sites in translational control are already widely used to maintain expression levels. In addition to current approaches, our framework allows a simultaneous multiple gene control. Furthermore, an inducible regulation that we offer, can make the search for perfect conditions a lot easier.

Species sign in ODE system	Species	Initial concentration (M)
A	pDNA+RNA I+RNAII early	0
B	pDNA+RNA II short	0
RNAI	RNA I	1E-6
D	pDNA+RNA II long	0
E	pDNA+RNAII primer	0
F	RNA II long	0
G	pDNA	4E-8*
H	pDNA+RNA II+RNA I late	0
RNA II	RNA II	0
J	RNAI+RNAII	0