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<h2> [[Part:BBa_K1934050|pTAC_CFP]]</a> characterization </h2>
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<h2> <a href"https://parts.igem.org/Part:BBa_K1934050">pTAC_CFP</a> characterization </h2>
  
 
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Revision as of 12:35, 20 October 2016


Ptac, trp & lac regulated promoter

The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. This part also exist together with lacI, part BBa_K180000

[1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

INSA Lyon 2016 Experiments on this part


pTAC_RFP characterization

Description

The pTAC promoter used corresponds to the part BBa_K864400. This promoter is a hybrid of two operons: the trp and lac operons.This promoter is inducible by IPTG and commonly used in Escherichia coli for overproduction of proteins. Escherichia coli NM522 strain that we used in our lab constitutively express the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed a strong leakage when plating our BBa_K1934000 transformants . Therefore, we decided to put a RFP reporter ORF under control of the pTAC promoter to characterize the promoter noise.

Expression of the pTAC-RFP fusion in presence of increasing amount of the inductor IPTG

Experimental design
The RFP coding sequence (BBa_E1010) was placed in silico under the control of the pTAC promoter (BBa_K864400), a strong RBS (BBa_B0030) and a bidirectional terminator (BBa_B0011). IDT realized the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 iGEM reference plasmid and then transformed into Escherichia coli NM522 strain. In order to study the efficiency of pTAC promoter for proteins overproduction, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions: 0 mmol.L-1, 1 mmol.L-1 and 5 mmol.L-1. The OD600 of each culture was measured each hour during six hours. Escherichia coli NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.
Results
  • Noise of the pTAC: fluorescence in absence of IPTG

We study the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.

Figure 1. Fluorescence/DO600 comparison between NM522 with or without the plasmid (RFP). Fluorescence of each sample was measured very hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; NM522 strain with plasmid reveals a higher protein expression.

An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.61, suggesting that the time have no effect on pTAC-RFP expression in absence of IPTG (α<0.05). Given this result, we can gather the data to analyse if there is a significant difference between the two strains. A Student test was performed with the variance not equal. The p-value of 5.44*10-4 indicates that the strain carrying pTAC-RFP transcriptional fusion display a higher fluorescence than the control strain.

  • pTAC induction by increasing concentration of IPTG

We now study the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L-1.

Figure 2. Fluorescence/DO600 comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 0 or 1 mmol.L-1. Fluorescence of each sample was measured very hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; IPTG induction enables a higher protein expression rate.

An ANOVA test was made to see if there was a time effect between the two populations. The p-value of 0.21 indicates that the time have no effect on the fluorescence induction (α<0.05). The data were therefore gathered in order to compare the strain fluorescence with 1 mmol.L-1 IPTG and without. We realize a Student test with the variance not equal and obtain a p-value of 8.57*10-3, showing a difference of fluorescence due to the presence of IPTG in the medium.

Finally, we compared the expression of the pTAC-RFP transcriptional fusion in 2 concentrations of IPTG: 1 and 5 mmol.L-1.

Figure 3. Fluorescence/DO600 comparison between NM522 strains with plasmid (RFP) and with IPTG induction of 1 or 5 mmol.L-1 Fluorescence of each sample was measured very hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. Even after six hours of incubation, a significant expression difference can't be detected, the protein expression rate is not correlated with the concentration of IPTG.

An ANOVA was made to see if there was a time effect between the two populations. The p-value of 0.06 indicates that the time have no effect (α<0.05). From there, we can gather the data to analyse if there is a significant difference between the two concentrations of IPTG.

We realize a Student test with the variance not equal and obtain a p-value of 0.61, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.

Conclusion

In the case of RFP, the pTAC promoter seems to not enable a gene tune because statistics show that there is a significant noise, even in absence of IPTG.

pTAC_CFP characterization

Description

The pTAC promoter used corresponds to the part BBa_K864400. This promoter is a hybrid of two operons: the trp and lac operons.This promoter is inducible by IPTG and commonly used in Escherichia coli for overproduction of proteins. Escherichia coli NM522 strain that we used in our lab constitutively express the LacIq protein, a strong pTAC promoter repressor. However, in absence of IPTG, we observed a strong leakage when plating our BBa_K1934000 transformants. Therefore, we decided to put a CFP reporter ORF under control of the pTAC promoter to characterize the promoter noise.

Expression of the pTAC-CFP fusion in presence of increasing amount of the inductor IPTG

Experimental design
The CFP coding sequence (BBa_E2020)(BBa_E2020) was placed in silico under the control of the pTAC promoter (BBa_K864400), a strong RBS (BBa_B0030) and a bidirectional terminator (BBa_B0011). IDT realized the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into pSB1C3 iGEM reference plasmid and then transformed into Escherichia coli NM522 strain. In order to study the efficiency of pTAC promoter for proteins overproduction, recombinant clones were grown overnight in LB at 37°C in duplicate in three different induction conditions: 0 mmol.L-1, 1 mmol.L-1 and 5 mmol.L-1. The OD600 of each culture was measured each hour during six hours. Escherichia coli NM522 strain was grown overnight in LB at 37°C in the same three induction conditions as control.
Results
  • Noise of the pTAC: fluorescence in absence of IPTG

We study the noise of the promoter by comparing the normalized fluorescence between the construction and the NM522 strain without any induction of IPTG.

Figure 4. Fluorescence/DO600 comparison between NM522 with or without the plasmid (CFP). Fluorescence of each sample was measured very hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; NM522 strain with plasmid reveals a higher protein expression.

An ANOVA was made to see if there was a time effect between the two populations. We obtain a p-value of 0.96, suggesting that the time have no effect on pTAC-CFP expression in absence of IPTG (α<0.05). Given this result, we can gather the data to analyse if there is a significant difference between the two strains. A Student test was performed with the variance not equal. The p-value of 0.10 indicates that the strain carrying pTAC-CFP transcriptional fusion doesn’t display a significant fluorescence difference with the control strain.

  • pTAC induction by increasing concentration of IPTG

We now study the induction of the promoter by comparing the normalized fluorescence of the construction under the induction of [IPTG] = 0 and 1 mmol.L-1.

Figure 5. Fluorescence/DO600 comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 0 or 1 mmol.L-1. Fluorescence of each sample was measured very hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. After one hour, a significant expression difference is remarkable; IPTG induction enables a higher protein expression rate.

An ANOVA test was made to see if there was a time effect between the two populations. The p-value of 0.05 indicates that the time have no effect on the fluorescence induction (α<0.05). The data were therefore gathered in order to compare the strain fluorescence with 1 mmol.L-1 IPTG and without. We realize a Student test with the variance not equal and obtain a p-value of 4.64*10-10, showing a significant difference of fluorescence due to the presence of IPTG in the medium.

Finally, we compared the expression of the pTAC-CFP transcriptional fusion in 2 concentrations of IPTG: 1 and 5 mmol.L-1.

Figure 6. Fluorescence/DO600 comparison between NM522 strains with plasmid (CFP) and with IPTG induction of 1 or 5 mmol.L-1 Fluorescence of each sample was measured very hour during six hours with ChemiDoc image system and was normalized by dividing by the OD600. Even after six hours of incubation, a significant expression difference can't be detected, the protein expression rate is not correlated with the concentration of IPTG.

An ANOVA was made to see if there was a time effect between the two populations. The p-value of 0.55 indicates that the time have no effect (α<0.05). From there, we can gather the data to analyse if there is a significant difference between the two concentrations of IPTG. We realize a Student test with the variance not equal and obtain a p-value of 0.54, indicating that no significant difference of fluorescence was observed with the rise of IPTG concentration.

Conclusion

In the case of CFP, the pTAC promoter seems to enable a gene tune because there isn’t a differential gene expression in absence of IPTG, so a significant noise isn’t measured.