OVERVIEW

The goal of Interlab is to establish a GFP measurement protocol based on engineering principles that anyone with a plate reader can use in their lab.Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology.However, the ability to repeat measurements in different labs has been difficult. So ,through INTERLAB every team use the same exact protocol around the world to produce common, comparable units for measuring GFP with different plate readers.

This year,interlab has provided a standard protocol for making measurements in a plate readeror a flow cytometer. We are going to test some Regulatory and RBS devices (BCDs) that are intended to make gene expression more precise and reliable. All biobricks to be tested have been sent in plasmid form. An excel sheet has been provided, to facilitate ease of data processing.

PARTS

we are provided with the eight plasmids containing the composite parts we will need for the InterLabStudy.These plasmids include six test devices,along with two controls.For the six test devices,the GFP (E0040) and the terminator (B0010,BOO12) are common. They differ in promotor, which are J23101 ,J23106,J23117.The negtive control only has RBS(R0040).The positive control has other Promotor(J23151) and terminator(B0032).The strength of the promotor or RBS can be then compared as it is directly proportional to the amount of fluorescence.

table.1 parts of 8 biobricks

Table2.variant RFP（au）

PROTOCOL

1.Making competent cells http://parts.igem.org/Help:Protocols/Competent_Cells

We tried twice to make competent cells with high transformation efficiency.

Fig.1 1μL of 100pg/μL BBa-J04450 to transform the competent cells we made

2.Resuspension of plasmid DNA

Resuspended DNA Distribution Kit well with 10μL dH2O.Pipet up and down several times,sit for a few minutes and the resuspension turned red.

3.Transformation

http://parts.igem.org/Help:Protocols/Transformation

We did transformation four times because device1 was too difficult to transform.We got only one colony for the first three transformation.For the last transformation, we extended the heat shock to 90 seconds,so we got great colonies of device1.Store all plates at 4℃ after picking single colony.

Fig.2 device1 culture after extending heat shock time to 90s

4.Colony PCR

Pick a single colony from each plate and do a colony PCR to verify part size.Grow up cell cultures thenadd 30%glycerin to the ratio of 1 to 1 before storing at ﹣80℃.

Table3.PCR system and method

Fig3. colony PCR product of 16 colonys

After colony PCR,sesequenced the colonies,and the results were positive.

5.Sampling

https://2017.igem.org/Competition/InterLab_Study/Plate_Reader

We chose plate reader for sampling and set Excitation of 485nm, Emission 540nm，the rest is the same as the method in the line.

Result (check out here)

We found that the promoter have a great effect on the expression of GFP, whereas RBS had little effect on GFP.Interestingly,the E.coli with high expression of GFP have low OD value.

fig4.the fluorescence intensity of 16 colony

Fig5.the growth curve of 16 colonys