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Project Hunger

Introduction

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.

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 cell has sufficient NtrC-P when the concentration of ammoniacal source is low.

Fig.2 Cycles of NtrB and NtrC

The concentration of ammoniacal source is detected by the ratio of αketoglutarate to glutamine. If glutamine levels are low, less αketoglutarate 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.

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

Although this nitrogen controlled switch is complex,

However, the σ⁵⁴promoter has never been evaluated quantitatively. We characterize 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 following reasons:

• We know that it is a lot of trouble to calculate RPU using GFP fluorescence without platereader.
• Because the σ⁵⁴promoter relates to the metabolism of glutamine, the concentration of the glutamine changes and activity of σ⁵⁴promoter changes.

When we calculate RPU using GFP fluorescence, we need to measure GFP fluorescence at two points in an exponential growth phase and 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.

† NtrB and NtrC are otherwise called NR_II, NR_I

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.

This time we intended to assay the activity of promoter against glutamine concentration. It is desirable to induct after the cultivation in the medium without glutamine. However, from our last year's experience, we knew that E.coli grew really inefficiently in a M9 medium without casamino acids. So, at first we cultivated E.coli in the medium with casamino acids overnight, then changed the medium with the one without casamino acids. After that, we performed an experiment to determine how long it took to reach a steady state.

When we use new way to measure RPU, we need to know the length of time to reach steady state. So, we made the following preliminary experiments to measure it.

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 extracted RNA and synthesized cDNA. Finally, we used real time PCR.

Result

We get this new equation for measuring RPU.

The derivation is below.

RPU is defined as follows.

(1)

PoPS (Polymerase Per Second) is the unit of absolute “promoter activity”. It is defined as the number of RNA polymerase molecules that pass by the final base pair of the promoter and continue along DNA as an elongation complex.

(2)

Where:

• [mRNA] is the concentration of mRNA,
• γ is the mRNA degradation rate,
• n is the copy number of the plasmid containing the promoter

Following equations related to promoterφ and J23101 are derived because d[mRNA]/dt = 0.

(3)

(4)

If the test promoter φ and the reference standard promoter are measured under the same culture conditions and both promoters are carried on the same backbone plasmid, following equations are approved.

(5)

(6)

Equation(3) divided by (4) is (7) .

(7)

We can reduce (7) because of (5) and (6) and use (1). Then we get this equation.

(8)

So, we can calculate RPU with this equation.

Following graphs are the results of real time PCR.

Fig.1 :graph1. the relative amount of RNA expression

Fig.2 :graph2. the relative amount of RNA expression except TBP

Fig.3 :graph3. RNA expression corrected by PGK

Fig.4 :graph4. RNA expression corrected by TBP

Fig.5 :graph5. RNA expression corrected by GAPDH

Fig.6 :graph6. RNA expression corrected by actin