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Latest revision as of 03:56, 6 October 2011

Project Hunger

Carnivorous E.coli attracts insects by emitting light, but it is a burden for the E.coli. To reduce this burden, we use nitrogen regulatory proteins, NtrB and NtrC. They activate σ54 promoter when the supply of nitrogen is not enough. NtrB and NtrC are coded in glnL and glnG, respectively.

† NtrB and NtrC are otherwise called NR_II, NR_I

Introduction

Mechanism of Regulation of Transcription Depending on the Concentration of Glutamine

Ammonia is an essential nitrogen source for the bactria. When enteric bacteria are deprived of ammonia, they express glnA to produce glutamine synthetase(GS) under the σ⁵⁴promoter. The transcription from σ⁵⁴promoter is stimilated by phosphorylated form of NtrC(NtrC-P). The σ⁵⁴ RNA polymerase binds to the glnA promoter, forming a closed complex, but cannot form an open complex and initiate transcription until it is activated by NtrC-P. NtrC is phosphorylated by NtrB-P, an autokinase which phosphorylates itself with ATPs. Phosphorylation and dephosphorylation of NtrB and C are controlled so that a cell has sufficient NtrC-P when the concentration of ammoniacal source is low.

Fig.1 Regulation of σ⁵⁴promoter

Fig.2 Cycles of NtrB and NtrC

The concentration of ammoniacal source is detected by the ratio of α-ketoglutarate to glutamine. If glutamine levels are low, less α-ketoglutarate is synthesized by GS and, as a result, Pii retains UMP and so cannot bind to NtrB. NtrB can then phosphorylate itself and transfer this phosphate to NtrC.

Fig.3 Detection of Ammoniacal Source

[GS Reaction Under Low NH3 Concentration]
Glutamate + NH3 + ADP  →  Glutamine + ADP + phosphate
                      GS
Glutamine + αketoglutarate  →  2 glutamate
                            GS

In our project, we decided to use NtrB and NtrC to control the expression level of genes for the following reasons:

• They were not submitted to the partsregistry.
• Many researches have been done on NtrB and NtrC, while there are not as many reports on Pii and GS.
• The cell signalling mechanism can be made simple.
• σ⁵⁴promoter was not quantitatively characterized.

We characterized the σ⁵⁴promoter by Relative Promoter Unit (RPU), because absolute promoter activity depends on test conditions and measuring instruments. RPU can reduce this Coefficient of Variation (CV) from 39.1% to 17.5% [2]. Therefore RPU can make it easier for us to share the data of promoter activity and use BioBricks.

We have used GFP fluorescence to measure RPU previously, but this time, we tried to calculate RPU using another way for the reasons below:

• We know that it is a lot of trouble to calculate RPU using GFP fluorescence without platereader.
• As the σ⁵⁴promoter relates to the metabolism of glutamine, the change in the concentration of glutamine results into the change of activity of σ⁵⁴promoter.

When we calculate RPU using GFP fluorescence, we need to measure GFP fluorescence at two points in an exponential growth phase and on the same glutamine concentration, but we have the following problems.

• before E.coli reach exponential growth phase, the concentration of glutamine changes
• the concentration of glutamine is different between one point and the other point.

We devised the new way of calculating RPU using the amount of mRNA, according to the following equation.

We can characterize promoters with RPU easily by using this new way.

Method

We created the following two constructions to characterize σ⁵⁴ promoter with RPU depending on the concentration of glutamine. One construction includes BBa_J23101 as a promoter which is used as the standard and the other includes binding sites and σ⁵⁴ promoter.

Result

New equation for measurement RPU

We get this new equation for measuring RPU.(in steady state)

(To see the derivation, see Team:Kyoto/Hunger/Modeling.)

How long E.coli can keep steady state after transferred to medium lack of nutrition

To measure RPU by our new method, we need to know how long E.coli can keep steady state after transferred to medium lack of nutrition. We think that cell population and gene expression keep in steady state. So we measured OD600 and expression of stationary phase specific gene, csiE and bolA.

In order to measure the RPU of σ⁵⁴promoter depending on glutamine concentration, the cellular density of mRNA must be measured when it is constant(this means that the concentration of glutamine is kept invariable). So, E.coli must be in the steady state. However, E.coli consumes glutamine and it is impossible to add glutamine at the first point of cultivation. Cultivation without any glutamine is necessary. Also, From our last year's experience, we knew that E.coli grew really inefficiently in M9 medium without glutamine, which is often suplied from the casamino acids. Thus, first we cultivated E.coli with casamino acids overnight, then exchanged the medium with that is free of casamino acids.

As a preliminary experiment of measurement of the RPU of σ⁵⁴promoter, we performed an experiment to determine how long it takes to reach a steady state.

We cultivated E.coli in M9 media(+ casamino acids) for about 15 hours and dispenced 2.4ml to each tubes. Then, we centrifuged these tubes (13,000 rpm , 4°C, 1min) and discarded the supernatant. We added 2.4ml media(- casamino acids) and centrifuged at 4°C twice. Again, we centrifuged these tubes and discarded the supernatant and added 2.4ml media(-casamino acids) at 37 °C. We brought up E.coli at 0,5,10,15,20,25,30,60min and mesured OD600 and extracted RNA and synthesized cDNA. Finally, we used real time PCR.

Following table is the result of OD600 in the preliminary experiment.

To use real time PCR, we need to choose internal control to correct data. We use GAPDH, TBP, PGK, and actin as candidate of internal control and research which gene is suitable to internal control.

Following graphs are the results of real time PCR in the preliminary experiment.

Fig.1 :graph1. the relative RNA expression level

This table is the relative RNA expression level used in graph1.

Fig.2 :graph2. the relative RNA expression level except TBP

This table is the relative RNA expression level except TBP used in graph2.

Fig.3 :graph3. the relative RNA expression level corrected by PGK

This table is the relative RNA expression level corrected by PGK used in graph3.

Fig.4 :graph4. the relative RNA expression level corrected by TBP

This table is the relative RNA expression level corrected by TBP used in graph4.

Fig.5 :graph5. the relative RNA expression level corrected by GAPDH

This table is the relative RNA expression level corrected by GAPDH used in graph5.

Fig.6 :graph6. the relative RNA expression level corrected by actin

This table is the relative RNA expression level corrected by actin used in graph6.

From these result, we chose GAPDH and actin as internal control. Next, we measured csiE and bolA, stationary phase specific gene, expression level to confirm whether 0~10 minutes after transferred is steady state.

Fig.7 :graph7. the csiE, stationary phase specific gene, expression level corrected by GAPDH or actin

This table is the csiE, stationary phase specific gene, expression level corrected by GAPDH or actin used in graph7.

Fig.8 :graph8. the bolA, stationary phase specific gene, expression level corrected by GAPDH or actin

This table is the bolA, stationary phase specific gene, expression level corrected by GAPDH or actin used in graph8.