Part:BBa_K2009430:Experience

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Results:

This is the picture which shows Escherichia coli (E.coli) containing the plasmid of sfGFP1-10 observed with excitation light.
This is the picture which shows E.coli containing the sfGFP11 plasmid and sfGFP1-10 plasmid observed with excitation light.
If you want to see all pictures, please go to our notebook.
experimental data:

sample	OD600	ABS
A10-1	2.541	8402
A10-2	1.486	5389
C11-1	2.539	9939
C11-2	2.230	8098
A+C-1	1.162	3078
A+C-2	1.077	2627

The ideal results are the spilt GFP experiment in E-coli to verify the split GFP can function as our expectation, we transform the plamids containing sfGFP1-10 and the plasmids containing sfGFP11 respectively in BL21, cultivating the bacteria at 37°C and shacking at 250 rpm/min overnight. We use fluorescence microscope to observe bacteria under 100X objective lens. From these fluorescent images, we find that fluorescent intensity of sfGFP1-10 is stronger than fluorescent intensity of sfGFP11 and both of them are weak. It corresponds with our expectation that either of separate part of sfGFP have background expression and sfGFP1-10, which is longer, may be brighter after excitation.

However, when the PSB1C3 carrying the part of sfGFP11 and the PSB1A3 carrying the part of sfGFP1-10 ware expressed together in E.Coli, it doesn't present stronger fluorescence intensity than either plasmid is expressed in E.Coli respectively. There are a few reasons can explain it. Firstly, different metabolic stress of two plamids causes indistinct results. PSB1C3 carrying sfGFP11, whose gene length is shorter, may have higher expression level than PSB1A3 carry sfGFP1-10. Secondly, there is obvious fluorescence quenching after excitation. Therefore, results of fluorescent images and fluorescent images appear that co-expression of sfGFP1-10 and sfGFP11 has weaker fluorescence intensity. Thirdly, because sfGFP1-10 and sfGFP11 don’t express in E.Coli at 1:1 ratio, the collision probability may be lower than our expectation.