Difference between revisions of "Part:BBa K185004"

Revision as of 12:20, 1 November 2009

RelE toxin+Double terminator

It is composite part, which is composed by RelE toxin and double terminator.You will find the detailed information about RelE-toxin in BBa_K185047.

Sequence and Features

Characterization of RelE toxin+Double terminater(BBa_K185004)

It is a compsite part composed by BBa_K185047 and BBa_B0015. The creator of this part is Alex Jiang. When we insert different promoter ahead of this part, it will express RelE protein on different levels, depending on the strength of promoters. Its length is 443 bp, which can be verified by gel electrophoresis.

Fig 1. Construct results of RelE+Double terminator

• Lane2: BBa_K185000 RelE toxin+Rbs30 311bp
• Lane3: BBa_K185004 RelE toxin+Double terminator 443bp

The stop codon of wild type RelE gene is TGA. Here we mutated it into TAA, according to inhibition specifity towards different stop codons: UAG>UAA>UGA. The rates of the inhibition reactions in all cases were proportional to the relE concentration and thus determined by k_cat/K_m. The k_cat/K_m Values (s^-1μM^-1) for RelE Cleavage of 3 stop codons in the Ribosomal A Site have been measured previously. (UGA:0.078, UAA:2.2, UAG:26).

Fig 2. RelE inhibition of peptide release from ribosomes with a tetrapeptidyl-tRNA in the P site and a stop codon in the A site. From The Bacterial Toxin RELE Displays Codon-Specific Cleavage of mRNAs in the Ribosomal A site.

We have test this part under two different promoters: pLac and pBAD. After identification of reconstruced plasmid, we transfected the plasmid into E.coli BL21 strain and then incubated it into M9 medium. All the liquid M9 medium contains 0.1% IPTG and 0.1% Amp(In pBAD strain, 0.2% L-arabinose and 0.1% Amp), in order to keep the same growing conditions. The total amount of bateria is detected by spectrophotometer. It provides the real-time detecting data of bacteria amount under OD_600.

The growth data and fitting curve (according to logistic formulation) are demonstrated below:

Table 1 Variation of OD₆₀₀ Value with Time in Different Strains

Time(h)	OD(pLac+RelE)	OD(NC in IPTG)	OD(pBAD+RelE)	OD(NC in Arabinose)
0.000	0.0000	0.0000	0.0000	0.0000
1.000	0.0250	0.0100	0.0050	0.0250
2.000	0.0500	0.0250	0.0300	0.0200
3.000	0.0050	0.0100	0.0100	0.0150
4.250	0.0000	0.0150	0.0250	0.0350
5.000	0.0500	0.0100	0.0800	0.0500
6.000	0.1400	0.0350	0.1800	0.0950
7.000	0.2350	0.1500	0.3200	0.3850
8.000	0.2650	0.3350	0.3100	0.5100
8.500	0.2550	0.3150	0.3050	0.5100
9.000	0.2500	0.3250	0.3500	0.5950
9.500	0.2300	0.3600	0.4150	0.6350
10.500	0.3000	0.4150	0.4300	0.6450
11.500	0.3300	0.4550	0.4200	0.6950
12.500	0.2950	0.4900	0.4000	0.8050
13.500	0.2750	0.4800	0.4000	0.7000
14.500	0.2500	0.4600	0.3900	0.6750
16.000	0.2550	0.5050	0.4150	0.7100
18.000	0.2400	0.4700	0.4100	0.7650

Fig 3. IPTG-induced RelE strain growth curve

The green line represents the OD value of NC, whereas the red one represents RelE strain.

Fig 4. L-arabinose-induced RelE strain growth curve

The green line represents the OD value of NC, whereas the red one represents RelE strain.

The growth curve of both NC and RelE strain fit the typical bacteria growth pattern, including the lag phase, exponential phase, stationary phase, and death phase. However, the exponential phase (the most rapid phase in which bateria grows) in RelE strain shows a shorter period than in NC group. The shorter exponential period eventually results in lower concentration of bacteria in stationary phase. Consequensely, we can verify that expression of the relE gene has been shown to severely inhibit bacteria growth and prevent colony formation.