Difference between revisions of "Team:ITB INDONESIA/results/lab"

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<h2>Lab Results</h2>
 
<h2>Lab Results</h2>
 
<section>
 
<section>
−
<p>
+
<p>We utilise The Biosurfactor biobrick (BBa_K653000) designed by Panama 2011 that encodes rhlA and rhlB, two enzymes for rhamnolipid production. We put the biobrick under T7lac promoter with strong RBS (BBa_K613010), and double terminator BBa_B0015. We order our part to be chemically synthesised via BioBasic.</p>
−
We utilise The Biosurfactor biobrick (BBa_K653000) designed by Panama 2011 that encodes rhlA and rhlB, two enzymes for rhamnolipid production. We put the biobrick under T7lac promoter with strong RBS (BBa_K613010), and double terminator BBa_B0015.</p>
+
 
−
<p>
+
<p>We transformed the part in BioBasic plasmid into E. coli DH5a. The confirmed plasmid was then used for transformation into E. coli BL21(DE3). The true transformant was used for expression.</p>
−
We designed our Rhamncolipid to express reporter protein when producing rhamnolipid. We tried to find best reporter for our system that has fast turnover and doesn’t have significant fitness cost to the bacteria. We tested two reporters, RFP and aeBlue, with each reporter has different tags, either not tagged, LAA-tagged, or LVA-tagged.</p>
+
 
−
<p>
+
<p>We assembled out part in the BioBasic plasmid into pSB1C3 encoding RFP and then transformed it into E. coli DH5a. We used 2 ligation composition, and transformed each mix. Utilising red-white colony selection, we selected 4 white colonies candidate for colony PCR from each plate. Four of our selected colonies showed positive result: having a band around 2500-3000 kb.</p>
−
<p>We also designed our control device which encodes LacI under different promoter strength, strong, medium, or even without the control.</p>
+
 
−
<h3>Reporter Selection</h3>
+
<div class="img-caption">
−
<p>We transformed E. coli BL21(DE3) with different reporter proteins (RFP and aeBlue) with different tags (none, LAA, LVA). We induced the transformants using IPTG and observe the OD600 for bacteria growth and measure each reporter.</p>
+
<img src="https://static.igem.org/mediawiki/2015/a/a4/ITB_INDONESIA_IGEM2015_PM_Verif2.png">
−
<p>The results showed that [to be continued]. Therefore, we selected [to be continued] as our reporter device.</p>
+
<span></span>
 +
</div>
 +
 
 +
<p>It was further confirmed by plasmid isolation and PCR, showing that we have successfully clone our part into pSB1C3</p>
 +
 
 +
 
 +
<div class="img-caption">
 +
<img src="https://static.igem.org/mediawiki/2015/9/93/ITB_INDONESIA_IGEM2015_PM_Verif.png">
 +
<span></span>
 +
</div>
 +
 
 +
<p>We designed our Rhamcolipid to express reporter protein when producing rhamnolipid. We tried to find best reporter for our system that has fast turnover and doesn’t have significant fitness cost to the bacteria. We tested two reporters, RFP and aeBlue, with each reporter has different tags, either not tagged, LAA-tagged, or LVA-tagged. We also designed our control device which encodes LacI under different promoter strength, strong, medium, or even without the control. But unfortunately to run out of time to assemble these devices.</p>
 +
 
 
<h3>Rhamnolipid production</h3>
 
<h3>Rhamnolipid production</h3>
−
<p>We transformed E. coli BL21(DE3) with rhamnolipid production module. After IPTG induction, the culture was centrifuged. The cell was collected and lysed for protein analysis by SDS-PAGE. The supernatant was collected and tested for surfactant activity.</p>
+
<p>We transformed E. coli BL21(DE3) with rhamnolipid production module. After IPTG induction, the culture was centrifuged. The cell was collected and lysed for protein analysis by SDS-PAGE. It showed that there is a band that we assumed to be our rhlA and rhlB due to their size around 30 and 40 kDa respectively. The low concentration of putative rhlA and rhlB might be caused by the codon usage. But it needs more confirmation and further studies.</p>
 +
 
 +
<div class="img-caption">
 +
<img src="https://static.igem.org/mediawiki/2015/d/d7/ITB_INDONESIA_IGEM2015_SDS_Page.png">
 +
<span></span>
 +
</div>
 +
 
 +
<p>The supernatant was collected and tested for surfactant activity.</p>
 +
 
 
<h3>Rhamnolipid test</h3>
 
<h3>Rhamnolipid test</h3>
−
<p>We tested our produced rhamnolipid for its characteristics and surfactant activites.</p>
+
We tested our produced rhamnolipid for its characteristics and surfactant activites. Here we describe for the very first time the usage of
−
<p>The results showed that supernatant from empty cells, induced or uninduced, and uninduced transformants had no or little surfactant activity. Supernatant from induced transformant has surfactant activity, comparable to synthetic surfactant, Tween 20%.</p>
+
The emulsification result showed that our supernatant has similar feature with positive control, Tween 20%, while our LB media (not from transformant’s supernatant) had similar property with negative control, water.
 +
 
 +
<div class="img-caption">
 +
<img src="https://static.igem.org/mediawiki/2015/b/b8/ITB_INDONESIA_IGEM2015_Emulsification.png">
 +
<span></span>
 +
</div>
 +
 
 +
<p>Using oil drop test, it was showed that supernatant from empty cells, induced (EI) or uninduced (EU), and uninduced transformants (TU) had no or little surfactant activity. Supernatant from induced transformant (TI) has surfactant activity, even though not as high as synthetic surfactant, Tween 80%. We tried different volume (10-30 uL) of the tested supernatant in 20 uL of crude oil.</p>
 +
 
 +
<div class="img-caption">
 +
<img src="https://static.igem.org/mediawiki/2015/9/91/ITB_INDONESIA_IGEM2015_lab_graphic.png">
 +
<span></span>
 +
</div>
 +
 
 +
<p>We tested the supernatant in Lemigas, Indonesia’s national research and development centre for oil and gas technology. We tested the supernatant’s interfacial tension (IFT) in 60<sup>o</sup>C and 70<sup>o</sup>C. It is showed that the supernatant from induced transformant has lower IFT (which is better) in 60<sup>o</sup>C. This IFT was even lower in higher temperature, 70<sup>o</sup>C. The IFT was within the range of good quality surfactant. Therefore, they were interested to do more test, but we did not have enough time to receive the result before Wiki Freeze.</p>
 +
 
 +
<div class="img-caption">
 +
<img src="https://static.igem.org/mediawiki/2015/f/ff/ITB_INDONESIA_IGEM2015_lab_graphic2.png">
 +
<span></span>
 +
</div>
 +
 
 
</section>
 
</section>
 
</html>
 
</html>
 
{{ITB_INDONESIA/footer}}
 
{{ITB_INDONESIA/footer}}

Revision as of 22:29, 17 September 2015

Lab Results

We utilise The Biosurfactor biobrick (BBa_K653000) designed by Panama 2011 that encodes rhlA and rhlB, two enzymes for rhamnolipid production. We put the biobrick under T7lac promoter with strong RBS (BBa_K613010), and double terminator BBa_B0015. We order our part to be chemically synthesised via BioBasic.

We transformed the part in BioBasic plasmid into E. coli DH5a. The confirmed plasmid was then used for transformation into E. coli BL21(DE3). The true transformant was used for expression.

We assembled out part in the BioBasic plasmid into pSB1C3 encoding RFP and then transformed it into E. coli DH5a. We used 2 ligation composition, and transformed each mix. Utilising red-white colony selection, we selected 4 white colonies candidate for colony PCR from each plate. Four of our selected colonies showed positive result: having a band around 2500-3000 kb.

It was further confirmed by plasmid isolation and PCR, showing that we have successfully clone our part into pSB1C3

We designed our Rhamcolipid to express reporter protein when producing rhamnolipid. We tried to find best reporter for our system that has fast turnover and doesn’t have significant fitness cost to the bacteria. We tested two reporters, RFP and aeBlue, with each reporter has different tags, either not tagged, LAA-tagged, or LVA-tagged. We also designed our control device which encodes LacI under different promoter strength, strong, medium, or even without the control. But unfortunately to run out of time to assemble these devices.

Rhamnolipid production

We transformed E. coli BL21(DE3) with rhamnolipid production module. After IPTG induction, the culture was centrifuged. The cell was collected and lysed for protein analysis by SDS-PAGE. It showed that there is a band that we assumed to be our rhlA and rhlB due to their size around 30 and 40 kDa respectively. The low concentration of putative rhlA and rhlB might be caused by the codon usage. But it needs more confirmation and further studies.

The supernatant was collected and tested for surfactant activity.

Rhamnolipid test

We tested our produced rhamnolipid for its characteristics and surfactant activites. Here we describe for the very first time the usage of The emulsification result showed that our supernatant has similar feature with positive control, Tween 20%, while our LB media (not from transformant’s supernatant) had similar property with negative control, water.

Using oil drop test, it was showed that supernatant from empty cells, induced (EI) or uninduced (EU), and uninduced transformants (TU) had no or little surfactant activity. Supernatant from induced transformant (TI) has surfactant activity, even though not as high as synthetic surfactant, Tween 80%. We tried different volume (10-30 uL) of the tested supernatant in 20 uL of crude oil.

We tested the supernatant in Lemigas, Indonesia’s national research and development centre for oil and gas technology. We tested the supernatant’s interfacial tension (IFT) in 60oC and 70oC. It is showed that the supernatant from induced transformant has lower IFT (which is better) in 60oC. This IFT was even lower in higher temperature, 70oC. The IFT was within the range of good quality surfactant. Therefore, they were interested to do more test, but we did not have enough time to receive the result before Wiki Freeze.

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