Team:Warsaw/RBSmeasurement

General info

In order to fine-tune expression of genes used in our project we have conducted measurement of various ribosome binding sites included in 2011 spring distribution. Our list of measured parts includes YFP E0030 (part 723 bp pSB1AK3), GFP E0020 (part 723bp pSB1A2), RFP E1010 (part 681 bp pSB2K3), mOrange E2050, MATSG—GSGTA linkers (part 744bp pSB2K3), GFP E0040 GFPmut3b (720 bp pSB1A2). Also DNA of SUPERFOLDER GFP, which was included in this distribution as a bigger part with RBS and promotor. We obtained coding sequence of SUPERFOLDER GFP using PCR method.

Sample preparation

To measure fluorescence of samples 3 milliliters of LB with AMP were inoculated with a colony from the plate. Cultures were vigorously shaken in 37° C overnight. The next step was to centrifuge 1,5 milliliters of liquid culture in an eppendorf tube. Pellet was then resuspended in 100 microliters of RF. Finally, prepared samples were applied on the fluorimeter plates. We used Corning 96 deep well plates with clear UV transparent bottom. Measurments were carried out using Tecan HS2000 pro fluorimeter with bottom read mode and using active gain optimization function. Wavlength values for individual proteins:

	GFP	YFP	RFP	mORANGE	SF-GFP
Excitation [nm]	488	514	554	545	488
Emission [nm]	509	530	585	568	508

We had also found our measure depend on antibiotics resistance. We measured two of samples 3 ml of LB supplemented with chloramphenicol was inoculated with a single colony and incubated at 37oC with shaking. The charts below show the results of measuring fluorescence.