Difference between revisions of "Part:BBa K2505030"
 
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<partinfo>BBa_K2505030 parameters</partinfo>
 
<partinfo>BBa_K2505030 parameters</partinfo>
 
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The gene <i>traI</i>(K34G) is derived from <i>Agrobacterium tumefaciens</i> and encode a enzyme necessary for synthesizing Quorum Sensing signaling molecules ([N-]acyl-homoserine lactones, AHLs), 3OC8 HSL (hereafter C8), in <i>E. coli</i>.
 +
This part constitutively produces C8. We introduced point mutation to <i>traI</i> gene and the productivity of C8 was improved by approximately 3-fold. We introduced this part to <i>E. coli</i> then <i>E. coli</i> could produced enough C8 to induce transcription of human cells.
 +
 +
The mutation was introduced wild type <i>traI</i>(<partinfo>BBa_K553001</partinfo>).
 +
 +
The DNA sequences of <i>traI</i> (K34G) is optimized for expressing in <i>E. coli</i> considering the codon usage.
  
−
This part constitutively produces C8. We introduced point mutation to <i>traI</i> gene, the productivity of C8 was improved by x fold. We introduced this part to <i>E. coli</i> then <i>E. coli</i> could produced enough C8 to induce transcription of human cells.
 
  
  
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==Characterization and improvement==
 
==Characterization and improvement==
−
In the previous wiki page (TraI Assay), we describe that the productivity of 3OC8HSL in E. coli heavily depends on the culture temperatures. However, to complete our co-culture system, the current C8 productivity at 37℃ was not enough to transmit the AHL signal to mammalian cells; note that mammalian cells are usually grown at 37℃. Therefore, we tried to mutate the traI gene and increase the productivity of 3OC8HSL at 37℃.
+
In our wiki page ([http://2017.igem.org/Team:TokyoTech/Experiment/TraI_ TraI Assay]), we describe that the productivity of 3OC8HSL in <span style="font-style: italic">E. coli</span> heavily depends on the culture temperatures. However, to complete our co-culture system, the current C8 productivity at 37℃ was not enough to transmit the AHL signal to mammalian cells; note that mammalian cells are usually grown at 37℃. Therefore, we tried to mutate the <span style="font-style: italic">traI</span> gene(<partinfo>BBa_K553001</partinfo>) and increase the productivity of 3OC8HSL at 37℃.
 +
<br>
 +
 
 +
[[File:T--TokyoTech--TraIimprove20.jpg|thumb|left|450px| '''Figure 1:''' '''Sequence of <i>traI</i> wild type gene and mutant. '''<br style="clear: both" />The mutations were introduced to the pSB1C3-based <i>traI</i> plasmid using the inverse-PCR method, and successful introduction of the mutations were confirmed with Sanger sequencing.]]
 +
TraI has not been characterized so extensively, and thus, it is unclear what kind of mutations is appropriate for the above purpose. A preceding study describes that, in the case of LuxI, the amino acid substitution at the 34th and 63rd positions (both are substitutions from glutamate to glycine; E34G and E63G) increase the productivity of C6 <sup>[1]</sup>. Since TraI has homology to LuxI over the entire amino acid sequences, we speculate that the same amino acid substitutions in TraI can increase the productivity of 3OC8HSL. The sequences of <i>traI</i> mutants and wild-type are shown in Figure 1.
 +
<br style="clear: both" />
 +
<br>
 +
[[File:T--TokyoTech--TraIimprove200.jpeg|thumb|left|450px| '''Figure 2:''' '''Structure of S-adenosylmethionine  ''']]In the experiments shown in this page, one additional modification was made in experimental conditions; 1 μM of SAM (S-adenosylmethionine; structure is shown in Figure 2) was added to the culture of the Sender. Since 3OC8HSL is synthesized from SAM and ACP (acyl carrier protein) through the action of TraI in bacterial cells <sup>[2]</sup>, we expected that the addition of SAM may increase the productivity.
 +
<br style="clear: both" />
 
==Result==
 
==Result==
−
[[File:https://static.igem.org/mediawiki/parts/0/0b/TraImutationResults.jpg|thumb|left|600px| '''Figure 1:''' '''3OC8HSL production of TraI wild type and mutant'''<br style="clear: both" />Sender E. coli were grown at 37℃ in liquid LB medium with 1μM of SAM. ]]<br>
+
[[File:TraImutationResults.jpg|thumb|left|450px| '''Figure 3:''' '''3OC8HSL production of TraI wild type and mutant'''<br style="clear: both" />Sender <i>E. coli</i>(producing TraI) were grown at 37℃ in liquid LB medium with 1μM of SAM. <i>E. coli</i> introduced empty vector was used as Negative Control.]]<br>
 +
The result of C8 production using the wild type TraI and mutants is shown in Figure 3.
 +
The RFU value of the TraI (K34G)-expressing cells was approximately 3-fold higher than that of the TraI-expressing cells. Other mutant didn’t show improvement of 3OC8HSL production. It was caluculated from calibration curve that the wild type TraI produced 28nM of C8 and TraI (K34G) produced 42nM of C8.
 +
 
 +
For more information, visit our page: [http://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement TraI_Improvement page].
  
−
The result of C8 production using the TraI wild-type and the mutants is shown in Figure 1.
 
−
The RFU value of the TraI-K34G-expressing cells was about 3-fold higher than that of the TraI-expressing cells. E. coli introduced empty vector was used as Negative Control.
 
−
Other mutant didn’t show improvement of 3OC8HSL production.
 
−
When these RFU values were converted to 3OC8HSL concentrations using the calibration curve obtained in the reagent assay (see the TraI Assay), they were calculated as 28 nM and 42 nM, respectively.
 
 
<br style="clear: both" />
 
<br style="clear: both" />
 
==Discussion==
 
==Discussion==
−
In the previous study, it was considered that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the acyl-ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect.  
+
In the previous study, it was showned that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the acyl-ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect. <br>
−
It was also showed that the MG1655hapB strain produced more C8 than the DH5α strain. We speculate that the difference in permeability of hydrophobic compounds through the cell membrane is the main reason for this result.
+
Taken together, we conclude that we could successfuly increase the productivity of C8. Notably, generation and functional identification the mutant TraI, TraI (K34G), meet the medal criteria of ”parts improvement”, because the wild-type <i>traI</i> parts was registered in iGEM parts collection earlier. However, further improvement of C8 production is necessary to transmit the signal from bacteria to mammalian cells. Such improvement is possible through tuning the experimental conditions further.
−
Taken together, we conclude that increasing the productivity of C8 at 37℃ was successful. Notably, generation and functional identification the mutant traI gene (TraI-K34G) meet the medal criteria of ”parts improvement”, because the wild-type traI parts was registered in iGEM parts collection earlier. However, further improvement of C8 production is necessary to transmit the signal from bacteria to mammalian cells. Such improvement is possible through tuning the experimental conditions further.
+
 
==Material and Method==
 
==Material and Method==
−
Supernatant assay
+
===Materials===
−
The procedure is the same as that described in the previous wiki page (TraI assay)
+
Strains<br>
−
except that (i) cells were cultured only at 37, (ii) 1 mM of SAM was added to the
+
Reporter<br>
−
culture medium at the step1, and (iii) two strains (DH5α, MG1655hapB) was used.
+
・DH5α<br>
 +
Sender E.coli<br>
 +
・DH5α<br>
 +
・MG1655hapB<br>
 +
Medium<br>
 +
・LB medium<br> containing 1μM of SAM<br>
 +
・LB medium containing antibiotics<br>
 +
Dissolve antibiotics in LB medium (Ampicillin 50μg/mL, Chloramphenicol 34μg/mL, Kanamycin
 +
50μg/mL)
 +
 
 +
===Method===
 +
Supernatant Assay <br>
 +
1. Grow the Sender in LB medium containing 1μM of SAM for about 15 hours at 37℃.<br>
 +
2. Harvest the cells by brief centrifugation<br>
 +
3. Perform the same procedure as the reagent assay process (1~4) to prepare Reporter culture.<br>
 +
4. Mix 250 μL of the supernatant of Sender culture with Reporter culture in a microtube.<br>
 +
5. Incubate the microtube for 5 hours with gentle shakingat 37℃.<br>
 +
6. Take 100 μL of the culture and measure fluorescence intensity (excitation and emission wave lengths
 +
are 495 and 520 nm, respectively) and turbidity (measurement wavelength is 600 nm).<br>
 +
7. Calculate RFU dividing fluorescence intensity by turbidity and minus control value.<br>
 +
 
 +
==Reference==
 +
[1] Pavan Kumar Reddy Kambam, Daniel J. Sayut, Yan Niu, Dawn T. Eriksen, Lianhong Sun (2008) Directed evolution of LuxI for enhanced OHHL production. Biotechnology and Bioengineering Volume 101, Issue 2 1 October 2008 Pages 263-272<br>
 +
 
 +
[2] MATTHEW R. PARSEK, DALE L. VAL, BRIAN L. HANZELKA, JOHN E. CRONAN, E. P. GREENBERG (1999) Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4360-4365, April 1999 Biochemistry<br>

Latest revision as of 11:01, 1 November 2017

Ptet-rbs-traI (K34G)-tt

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]


The gene traI(K34G) is derived from Agrobacterium tumefaciens and encode a enzyme necessary for synthesizing Quorum Sensing signaling molecules ([N-]acyl-homoserine lactones, AHLs), 3OC8 HSL (hereafter C8), in E. coli. This part constitutively produces C8. We introduced point mutation to traI gene and the productivity of C8 was improved by approximately 3-fold. We introduced this part to E. coli then E. coli could produced enough C8 to induce transcription of human cells.

The mutation was introduced wild type traI(BBa_K553001).

The DNA sequences of traI (K34G) is optimized for expressing in E. coli considering the codon usage.


Characterization and improvement

In our wiki page ([http://2017.igem.org/Team:TokyoTech/Experiment/TraI_ TraI Assay]), we describe that the productivity of 3OC8HSL in E. coli heavily depends on the culture temperatures. However, to complete our co-culture system, the current C8 productivity at 37℃ was not enough to transmit the AHL signal to mammalian cells; note that mammalian cells are usually grown at 37℃. Therefore, we tried to mutate the traI gene(BBa_K553001) and increase the productivity of 3OC8HSL at 37℃.

Figure 1: Sequence of traI wild type gene and mutant.
The mutations were introduced to the pSB1C3-based traI plasmid using the inverse-PCR method, and successful introduction of the mutations were confirmed with Sanger sequencing.

TraI has not been characterized so extensively, and thus, it is unclear what kind of mutations is appropriate for the above purpose. A preceding study describes that, in the case of LuxI, the amino acid substitution at the 34th and 63rd positions (both are substitutions from glutamate to glycine; E34G and E63G) increase the productivity of C6 [1]. Since TraI has homology to LuxI over the entire amino acid sequences, we speculate that the same amino acid substitutions in TraI can increase the productivity of 3OC8HSL. The sequences of traI mutants and wild-type are shown in Figure 1.

Figure 2: Structure of S-adenosylmethionine
In the experiments shown in this page, one additional modification was made in experimental conditions; 1 μM of SAM (S-adenosylmethionine; structure is shown in Figure 2) was added to the culture of the Sender. Since 3OC8HSL is synthesized from SAM and ACP (acyl carrier protein) through the action of TraI in bacterial cells [2], we expected that the addition of SAM may increase the productivity.


Result

Figure 3: 3OC8HSL production of TraI wild type and mutant
Sender E. coli(producing TraI) were grown at 37℃ in liquid LB medium with 1μM of SAM. E. coli introduced empty vector was used as Negative Control.

The result of C8 production using the wild type TraI and mutants is shown in Figure 3. The RFU value of the TraI (K34G)-expressing cells was approximately 3-fold higher than that of the TraI-expressing cells. Other mutant didn’t show improvement of 3OC8HSL production. It was caluculated from calibration curve that the wild type TraI produced 28nM of C8 and TraI (K34G) produced 42nM of C8.

For more information, visit our page: [http://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement TraI_Improvement page].


Discussion

In the previous study, it was showned that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the acyl-ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect.
Taken together, we conclude that we could successfuly increase the productivity of C8. Notably, generation and functional identification the mutant TraI, TraI (K34G), meet the medal criteria of ”parts improvement”, because the wild-type traI parts was registered in iGEM parts collection earlier. However, further improvement of C8 production is necessary to transmit the signal from bacteria to mammalian cells. Such improvement is possible through tuning the experimental conditions further.

Material and Method

Materials

Strains
Reporter
・DH5α
Sender E.coli
・DH5α
・MG1655hapB
Medium
・LB medium
containing 1μM of SAM
・LB medium containing antibiotics
Dissolve antibiotics in LB medium (Ampicillin 50μg/mL, Chloramphenicol 34μg/mL, Kanamycin 50μg/mL)

Method

Supernatant Assay
1. Grow the Sender in LB medium containing 1μM of SAM for about 15 hours at 37℃.
2. Harvest the cells by brief centrifugation
3. Perform the same procedure as the reagent assay process (1~4) to prepare Reporter culture.
4. Mix 250 μL of the supernatant of Sender culture with Reporter culture in a microtube.
5. Incubate the microtube for 5 hours with gentle shakingat 37℃.
6. Take 100 μL of the culture and measure fluorescence intensity (excitation and emission wave lengths are 495 and 520 nm, respectively) and turbidity (measurement wavelength is 600 nm).
7. Calculate RFU dividing fluorescence intensity by turbidity and minus control value.

Reference

[1] Pavan Kumar Reddy Kambam, Daniel J. Sayut, Yan Niu, Dawn T. Eriksen, Lianhong Sun (2008) Directed evolution of LuxI for enhanced OHHL production. Biotechnology and Bioengineering Volume 101, Issue 2 1 October 2008 Pages 263-272

[2] MATTHEW R. PARSEK, DALE L. VAL, BRIAN L. HANZELKA, JOHN E. CRONAN, E. P. GREENBERG (1999) Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4360-4365, April 1999 Biochemistry