Difference between revisions of "Part:BBa K2310100"
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<partinfo>BBa_K2310100 short</partinfo> | <partinfo>BBa_K2310100 short</partinfo> | ||
| − | Luminous bacteria are the most abundant and widely distributed of the light-emitting organisms and are found in marine, freshwater, and terrestrial environments. | + | Luminous bacteria are the most abundant and widely distributed of the light-emitting organisms and are found in marine, freshwater, and terrestrial environments. The most important feature of luminous bacteria which can produce the luciferase is called LuxAB, which can catalyzes the bioluminescence reactions. Almost all luminous bacteria have been classified into the three genera Vibrio, Photobacterium, and Xenorhabdus. In this part, the luciferase what we use is from the Xenorhabdus luminescens. |
[[Image:Structure-BBa_K2310100.gif|thumb|350px| 3D-Structure of luxAB luciferase]] | [[Image:Structure-BBa_K2310100.gif|thumb|350px| 3D-Structure of luxAB luciferase]] | ||
| − | LuxAB is a part of luxCDABEG which is the normal structure of the operon in most bioluminescent bacteria. The LuxCDE gene controls the synthesis/regenerate aldehyde and the | + | LuxAB is a part of luxCDABEG which is the normal structure of the operon in most bioluminescent bacteria. The LuxCDE gene controls the synthesis/regenerate aldehyde and the FMNH<sub>2</sub>, which is provided by an FMN reductase such as LuxG. The LuxAB luciferase is a heterodimeric enzyme of almost 80kDa composed of α and β-subunits whose molecular weight is 42kDa and 39kDa. For the two subunits, the α subunit plays a major role which is responsible for the light-emitting reaction and the β-subunit is important for stabling the protein, although there is about 40% identity in the amino acid sequence between the α and β subunits. |
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | As a luciferase, the light-emitting reaction which catalyzed by the LuxAB involves the oxidation of reduced riboflavin phosphate ( | + | As a luciferase, the light-emitting reaction which catalyzed by the LuxAB involves the oxidation of reduced riboflavin phosphate (FMNH<sub>2</sub>) and a long chain fatty aldehyde with the emission of blue-green light (490nm). This reaction is as follows: |
https://static.igem.org/mediawiki/parts/e/e6/M-luxab-BBa_K2310100.jpeg | https://static.igem.org/mediawiki/parts/e/e6/M-luxab-BBa_K2310100.jpeg | ||
| − | The reduced flavin, FMNH< | + | The reduced flavin, FMNH<sub>2</sub>, bound to the enzyme, reacts with O<sub>2</sub> to form a 4a-peroxyflavin. This complex interacts with aldehyde to form a highly stable intermediate, which decays slowly, resulting in the emission of light along with the oxidation of the substrates. |
| − | + | There are two ways to use the LuxAB as a reporting system, in heterologous hosts such as Escherichia coli. Either luxAB alone can be used (in which case decanal must be provided as substrate), or luxCDABE can be used, (in which case the organism can synthesize aldehyde itself). Because E.coli is capable for reducing the FMN to FMNH<sub>2</sub>, so it is not necessary to add luxG for E.coli host. | |
| − | + | ||
| − | There are two ways to use the LuxAB as a reporting system, in heterologous hosts such as Escherichia coli. Either luxAB alone can be used (in which case decanal must be provided as substrate), or luxCDABE can be used, (in which case the organism can synthesize aldehyde itself). Because E.coli is capable for reducing the FMN to FMNH, so it is not necessary to add luxG | + | |
In short, this luciferase can be used together with capraldehyde, producing luminescent and working as a reporter. | In short, this luciferase can be used together with capraldehyde, producing luminescent and working as a reporter. | ||
| Line 53: | Line 51: | ||
====Induction temperature==== | ====Induction temperature==== | ||
| − | We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter+luxAB (<partinfo>BBa_K2310103</partinfo>)in the fluid medium. When the value of OD600 reached about 1.2, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced at 30℃, 33℃ and 37℃, 190rpm shaking table for 3 hours respectively. Took 1ml bacteria solution in an EP tube, added 5ul substrate capraldehyde into EP tubes respectively and then took 100ulinto 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | + | We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter+luxAB (<partinfo>BBa_K2310103</partinfo>)in the fluid medium. When the value of OD600 reached about 1.2, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced at 30℃, 33℃ and 37℃, 190rpm shaking table for 3 hours respectively. Took 1ml bacteria solution in an EP tube, added 5ul substrate capraldehyde into EP tubes respectively and then took 100ulinto 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. We also did the experiment again under the same condition and induced in 30℃ and 37℃ for 3 hours. |
| − | + | <br><table><tr><th> | |
| − | + | [[Image:XMU-luxf1.png|thumb|400px|'''Figure 1''': Induction temperature –standard luminescence value]]</th><th> | |
| − | + | [[Image:XMU-luxf2.png|thumb|390px|'''Figure 2''': Induction temperature –standard luminescence value]]</th><th></table> | |
| − | Figure 1 | + | |
| − | + | ||
(Note: standard luminescence value= luminescence value-background value) | (Note: standard luminescence value= luminescence value-background value) | ||
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From the diagram, we can draw the conclusion that 30℃ is the best induction temperature among these three induction temperatures. | From the diagram, we can draw the conclusion that 30℃ is the best induction temperature among these three induction temperatures. | ||
| − | |||
====IPTG induction dosage==== | ====IPTG induction dosage==== | ||
We cultured the DH5a bacteria which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul, 8ul, 12ul, 16ul, 20ul IPTG in the tubes respectively. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | We cultured the DH5a bacteria which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul, 8ul, 12ul, 16ul, 20ul IPTG in the tubes respectively. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | ||
| + | <br><table><tr><th> | ||
| + | [[Image:XMU-luxf3.png|thumb|750px|'''Figure 3''': IPTG induction dosage-relative luminescence intensity]]</th><th></table> | ||
| − | |||
| − | |||
| − | |||
| − | |||
(Note1: relative luminescence intensity= luminescence value/background value) | (Note1: relative luminescence intensity= luminescence value/background value) | ||
| Line 87: | Line 70: | ||
| − | The diagram shows that when OD600 is low, the relative luminescence intensity will rise with the increase of the IPTG induction dosage within a certain range. However, when OD600 is high, the relative luminescence intensity may not rise with the increase of the IPTG induction dosage because the IPTG dosage is nearly saturated. By the way, 1.88 may be the best value of OD600 to test the influence of IPTG induction dosage. We guess that these two factors have an exponential function when OD600 is low; so we try to fit a curve under the first three values of OD600. | + | The diagram shows that when OD600 is low, the relative luminescence intensity will rise with the increase of the IPTG induction dosage within a certain range. However, when OD600 is high, the relative luminescence intensity may not rise with the increase of the IPTG induction dosage because the IPTG dosage is nearly saturated. By the way, 1.88 may be the best value of OD600 to test the influence of IPTG induction dosage. We guess that these two factors have an exponential function when OD600 is low; so we try to fit a curve under the first three values of OD600.<br><table><tr><th> |
| − | + | [[Image:XMU-luxf4.png|thumb|265px|'''Figure 4''': Induced at OD600=0.8]]</th><th> | |
| − | + | [[Image:XMU-luxf5.png|thumb|248px|'''Figure 5''': Induced at OD600=1.24]]</th><th> | |
| − | + | [[Image:XMU-luxf6.png|thumb|255px|'''Figure 6''': Induced at OD600=1.88]]</th></tr></table> | |
| − | + | ||
| − | Figure 4 | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Figure 5 | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Figure 6 | + | |
| − | + | ||
====Microbial concentration (OD600 value)==== | ====Microbial concentration (OD600 value)==== | ||
We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | ||
| − | + | <br><table><tr><th> | |
| − | + | [[Image:XMU-luxf7.png|thumb|750px|'''Figure 7''': OD600 value-relative luminescence intensity]]</th><th></table> | |
| − | + | ||
| − | Figure 7 | + | |
(Note: relative luminescence intensity= luminescence value/background value) | (Note: relative luminescence intensity= luminescence value/background value) | ||
| − | The diagram shows that the relative luminescence intensity will rise with the increase of OD600 value within a certain range. Besides, it seems that these two factors have an exponential function; as a result we try to fit a curve under different IPTG induction dosages. | + | The diagram shows that the relative luminescence intensity will rise with the increase of OD600 value within a certain range. Besides, it seems that these two factors have an exponential function; as a result we try to fit a curve under different IPTG induction dosages. <br><table><tr><th> |
| − | + | [[Image:Lux8.png|thumb|265px|'''Figure 8(a)''': OD600 value-relative luminescence intensity (1/k IPTG)]]</th><th> | |
| − | + | [[Image:XMU-luxf8b.png|thumb|265px|'''Figure 8(b)''': OD600 value-relative luminescence intensity (2/k IPTG)]]</th><th> | |
| − | + | [[Image:XMU-luxf8c.png|thumb|265px|'''Figure 8(c)''': OD600 value-relative luminescence intensity (3/k IPTG)]]</th></tr></table><br><table><tr><th> | |
| − | + | [[Image:XMU-luxf8d.png|thumb|265px|'''Figure 8(a)''': OD600 value-relative luminescence intensity (4/k IPTG)]]</th><th> | |
| − | Figure 8(a) | + | [[Image:XMU-luxf8e.png|thumb|265px|'''Figure 8(b)''': OD600 value-relative luminescence intensity (5/k IPTG)]]</th><th></th></tr></table> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Figure 8(b) | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Figure 8(c) | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Figure 8( | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | Figure 8( | + | |
When the concentration of IPTG is low, the exponential function between OD600 value and relative luminescence intensity is significant relatively. However, when the concentration of IPTG is high, the function is not significant. This is also because that the IPTG dosage is saturated relatively for the high OD600 value. | When the concentration of IPTG is low, the exponential function between OD600 value and relative luminescence intensity is significant relatively. However, when the concentration of IPTG is high, the function is not significant. This is also because that the IPTG dosage is saturated relatively for the high OD600 value. | ||
| − | |||
| − | |||
====Induction time==== | ====Induction time==== | ||
We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced1, 2, 3 hours in 30℃, 190rpm respectively, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced1, 2, 3 hours in 30℃, 190rpm respectively, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | ||
| − | + | <br><table><tr><th> | |
| − | + | [[Image:XMU-luxf9.png|thumb|750px|'''Figure 9''': Induction time-relative luminescence intensity]]</th><th></table> | |
| − | + | ||
| − | Figure | + | |
(Note1: relative luminescence intensity= luminescence value/background value) | (Note1: relative luminescence intensity= luminescence value/background value) | ||
| Line 161: | Line 105: | ||
This group of data is a bit odd; we cannot say the relationship between the induction time and relative luminescence intensity, so we tested the data in a different way. | This group of data is a bit odd; we cannot say the relationship between the induction time and relative luminescence intensity, so we tested the data in a different way. | ||
| + | <br><table><tr><th> | ||
| + | [[Image:XMU-luxf10.png|thumb|750px|'''Figure 10''': Induction time-relative luminescence intensity value]]</th><th></table> | ||
| − | |||
| − | |||
| − | |||
| − | |||
(Note1: relative luminescence value= (luminescence value-background value)/OD600 value after induction) | (Note1: relative luminescence value= (luminescence value-background value)/OD600 value after induction) | ||
| Line 172: | Line 114: | ||
| − | The diagram shows that the induction time has little influence on the relative luminescence value. Consequently, we can choose 1 hour as our induction time in order to improve the efficiency. | + | The diagram shows that the induction time has little influence on the relative luminescence value. Consequently, we can choose 1 hour as our induction time in order to improve the efficiency. |
| − | + | ||
====Substrate dosage==== | ====Substrate dosage==== | ||
We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteriasolution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 5ul, 10ul, 15ul, 20ul substrate capraldehyde into EP tubes respectively and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteriasolution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 5ul, 10ul, 15ul, 20ul substrate capraldehyde into EP tubes respectively and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. | ||
| − | + | <br><table><tr><th> | |
| − | + | [[Image:XMU-luxf11.png|thumb|750px|'''Figure 11''': Substrate dosage-relative luminescence intensity]]</th><th></table> | |
| − | + | ||
| − | Figure 11 | + | |
(Note1: relative luminescence intensity= luminescence value/background value) | (Note1: relative luminescence intensity= luminescence value/background value) | ||
Latest revision as of 16:07, 31 October 2017
LuxAB, emitting luciferase (from X. luminescens)
Luminous bacteria are the most abundant and widely distributed of the light-emitting organisms and are found in marine, freshwater, and terrestrial environments. The most important feature of luminous bacteria which can produce the luciferase is called LuxAB, which can catalyzes the bioluminescence reactions. Almost all luminous bacteria have been classified into the three genera Vibrio, Photobacterium, and Xenorhabdus. In this part, the luciferase what we use is from the Xenorhabdus luminescens.
LuxAB is a part of luxCDABEG which is the normal structure of the operon in most bioluminescent bacteria. The LuxCDE gene controls the synthesis/regenerate aldehyde and the FMNH2, which is provided by an FMN reductase such as LuxG. The LuxAB luciferase is a heterodimeric enzyme of almost 80kDa composed of α and β-subunits whose molecular weight is 42kDa and 39kDa. For the two subunits, the α subunit plays a major role which is responsible for the light-emitting reaction and the β-subunit is important for stabling the protein, although there is about 40% identity in the amino acid sequence between the α and β subunits.
Usage and Biology
As a luciferase, the light-emitting reaction which catalyzed by the LuxAB involves the oxidation of reduced riboflavin phosphate (FMNH2) and a long chain fatty aldehyde with the emission of blue-green light (490nm). This reaction is as follows:
The reduced flavin, FMNH2, bound to the enzyme, reacts with O2 to form a 4a-peroxyflavin. This complex interacts with aldehyde to form a highly stable intermediate, which decays slowly, resulting in the emission of light along with the oxidation of the substrates.
There are two ways to use the LuxAB as a reporting system, in heterologous hosts such as Escherichia coli. Either luxAB alone can be used (in which case decanal must be provided as substrate), or luxCDABE can be used, (in which case the organism can synthesize aldehyde itself). Because E.coli is capable for reducing the FMN to FMNH2, so it is not necessary to add luxG for E.coli host.
In short, this luciferase can be used together with capraldehyde, producing luminescent and working as a reporter.
Luminescent Wavelength
Substrate: Capraldehyde
Emission: 490nm
Protein data analysis
| Protein data table for LuxA automatically created by the BioBrick-AutoAnnotator version 1.0 | ||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Nucleotide sequence in RFC 10: (underlined part encodes the protein) ATGAAATTT ... CTATTATATTAG ORF from nucleotide position 1 to 1080 (excluding stop-codon) | ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
| ||||||||||||||||||||||||||||||||||||||||||||||
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid composition:
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid counting
| Biochemical parameters
| |||||||||||||||||||||||||||||||||||||||||||||
| Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges | ||||||||||||||||||||||||||||||||||||||||||||||
Codon usage
| ||||||||||||||||||||||||||||||||||||||||||||||
| The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment. | ||||||||||||||||||||||||||||||||||||||||||||||
| Protein data table for LuxB automatically created by the BioBrick-AutoAnnotator version 1.0 | ||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Nucleotide sequence in RFC 10: (underlined part encodes the protein) ATGAAATTT ... GAATATACCTAA ORF from nucleotide position 1 to 981 (excluding stop-codon) | ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
| ||||||||||||||||||||||||||||||||||||||||||||||
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid composition:
| ||||||||||||||||||||||||||||||||||||||||||||||
Amino acid counting
| Biochemical parameters
| |||||||||||||||||||||||||||||||||||||||||||||
| Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges | ||||||||||||||||||||||||||||||||||||||||||||||
Codon usage
| ||||||||||||||||||||||||||||||||||||||||||||||
| Alignments (obtained from PredictProtein.org) There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours. | ||||||||||||||||||||||||||||||||||||||||||||||
| Predictions (obtained from PredictProtein.org) | ||||||||||||||||||||||||||||||||||||||||||||||
| There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours. | ||||||||||||||||||||||||||||||||||||||||||||||
| The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment. | ||||||||||||||||||||||||||||||||||||||||||||||
Characterization
This BioBrick has been characterized. To see more information, you can visit the Experience page: https://parts.igem.org/Part:BBa_K2310100:Experience
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 530
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1049
Characterization of BBa_K2310100
Experimental objective
This experiment is designed to explore the influence of IPTG induction dosage, induction time, induction temperature, substrate dosage, microbial concentration (OD600 value), and other conditions to the chemiluminescence of luxAB luciferase.
Induction temperature
We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter+luxAB (BBa_K2310103)in the fluid medium. When the value of OD600 reached about 1.2, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced at 30℃, 33℃ and 37℃, 190rpm shaking table for 3 hours respectively. Took 1ml bacteria solution in an EP tube, added 5ul substrate capraldehyde into EP tubes respectively and then took 100ulinto 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. We also did the experiment again under the same condition and induced in 30℃ and 37℃ for 3 hours.
(Note: standard luminescence value= luminescence value-background value)
From the diagram, we can draw the conclusion that 30℃ is the best induction temperature among these three induction temperatures.
IPTG induction dosage
We cultured the DH5a bacteria which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul, 8ul, 12ul, 16ul, 20ul IPTG in the tubes respectively. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.
(Note1: relative luminescence intensity= luminescence value/background value)
(Note2: The relationship between the lines is incommensurable)
Microbial concentration (OD600 value)
We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.
(Note: relative luminescence intensity= luminescence value/background value)
When the concentration of IPTG is low, the exponential function between OD600 value and relative luminescence intensity is significant relatively. However, when the concentration of IPTG is high, the function is not significant. This is also because that the IPTG dosage is saturated relatively for the high OD600 value.
Induction time
We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced1, 2, 3 hours in 30℃, 190rpm respectively, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.
(Note1: relative luminescence intensity= luminescence value/background value)
(Note2: The relationship between the lines is incommensurable)
This group of data is a bit odd; we cannot say the relationship between the induction time and relative luminescence intensity, so we tested the data in a different way.
(Note1: relative luminescence value= (luminescence value-background value)/OD600 value after induction)
(Note2: The relationship between the lines is incommensurable)
The diagram shows that the induction time has little influence on the relative luminescence value. Consequently, we can choose 1 hour as our induction time in order to improve the efficiency.
Substrate dosage
We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteriasolution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 5ul, 10ul, 15ul, 20ul substrate capraldehyde into EP tubes respectively and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.
(Note1: relative luminescence intensity= luminescence value/background value)
(Note2: The relationship between the lines is incommensurable)
The diagram shows that when OD600 is low, the influence of substrate dosage on relative luminescence intensity is minimal. However, when OD600 is high, the relative luminescence intensity will rise with the increase of the substrate dosage within some certain range because of the accumulation of the luxAB luciferase.