Team:KULeuven/Data/GFP

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GFP with LVA-tag

Parts Registry:K145015

Introduction

Green fluorescent protein (GFP) is often used as a reporter protein, because it allows easy and nondestructive in situ monitoring of cellular processes. It can be expressed in a wide range of organisms and it doesn’t require the addition of a special substrate in order to detect green fluorescence. Nevertheless, the protein has one major drawback, it seems to be very stable. Once the expression of GFP is started, it will fluoresce for a very long period of time. This makes GFP unsuitable for monitoring rapid changes in gene expression. However, in our project fast changes of input will lead to fast changes of output. It is therefore of the utmost importance that we can easily monitor these changes. Andersen et al. [1] had already indicated that proper tagging of GFP will make the protein less stable. These researchers showed that a so-called LVA tag seemed to be the most efficient tag to make GFP unstable. This tag consists of a short peptide sequence (AANDENYALVA) and is attached to the C-terminal end of GFP. It is believed that the LVA tag renders GFP susceptible to the action of tail-specific proteases, namely Tsp protease in the periplasm and ClpXP and ClpAP in the cytoplasm of E.coli [2].

Since we have to monitor the variability of the gene expressions in our system, we decided to employ this fast degradable GFP mutant. We therefore transformed GFP with LVA tag into a BioBrick and characterized it with fluorescence-activated cell sorting (FACS).

Materials and methods

Media

The media used were Luria-Bertani (LB) medium or ABT minimal medium. LB medium was used to make liquid cultures (composition on OWW). ABT medium is a minimal AB medium (composition on OWW) containing 2.5 mg of thiamine per liter [1]. This ABT medium was used in our fluorescence measurements. Because ABT medium is a minimal medium, the production of new GFP stops and the degradation of GFP becomes detectable.

Constructing plasmids

First, we had to make a GFP with LVA tag in conformity to the BioBrick standard. Therefore we conducted a PCR with pfx polymerase on a plasmid containing the coding region of GFP-LVA that was given to us by Molin et al [1]. The primers that we used were built such that they would add a BioBrick prefix and suffix to the coding region of GFP-LVA (see below). This PCR product was then digested with EcoRI and SpeI. This digest was subsequently ligated into a pSB1A2 plasmid with T4 DNA ligase. Then we electroporated this ligation mix and obtained thus an E.coli strain containing K145015.

Now that we had a GFP-LVA BioBrick, we could construct a test module using the BioBrick Standard Assembly method. The test modules contain a promoter, RBS, coding region and terminator to allow the expression of GFP. We made the following constructs :

K145205	I7101

To make these constructs we miniprepped R0040, B0032, K14015, B0015 and E0240. After that R0040 was digested with EcoRI and SpeI, B0032 with EcoRI and XbaI, K145015 with PstI and XbaI, B0015 with SpeI and PstI, and E0240 with EcoRI and XbaI. We ligated some of these digests with T4 DNA polymerase : R0040+B0032, K145015+B0015 and R0040+E0240. These ligations were then electroporated into Top10 cells and the obtained colonies were miniprepped. R0040+B0032 was then cut with EcoRI and SpeI and K145015+B0015 was cut with EcoRI and XbaI. These digests were ligated to obtain part K145205 : (R0040+B0032)+(K145015+B0015). A schematic of this ligation procedure is shown below :

Measuring fluorescence of single cells

Electrocompetent Top10 cells were electroporated with K145205, I7101 and pUC18. These cells were plated out on agar plates with ampicillin and grown overnight (37°). From this, a 5ml liquid culture supplied with 100 µg ampicillin / ml was prepared and also grown overnight (37°C). These cells were inoculated in fresh LB medium (100 µg ampicillin / ml) in the morning and grown at 37°C for 3.5h. They were then washed in preheated (37°C) ABT minimal medium and resuspended in the 5ml of fresh ABT medium. After that, the cells were whithdrawn at different time intervals. They were diluted into PBS and analyzed by flow cytometry with a Becton Dickinson FACSCalibur and CELLQuest^TM acquisition and analysis software with gates set to forward and side scatters characteristic of the bacteria (Gate G2=R2).

Results and discussion

under construction...

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