Difference between revisions of "Team:Queens Canada/Improve"

Line 29: Line 29:
  
 
  <p><b>Previous Parts 4-HT Dependent Intein - </b><a href="http://parts.igem.org/Part:BBa_K2612001">http://parts.igem.org/Part:BBa_K2612001</a></p>
 
  <p><b>Previous Parts 4-HT Dependent Intein - </b><a href="http://parts.igem.org/Part:BBa_K2612001">http://parts.igem.org/Part:BBa_K2612001</a></p>
−
     <p> We tested the ability of the 4-hydroxytamoxifen (4-HT) dependent intein to splice in bacteria, rather than yeast in attempts to improve its characterization, and with the ultimate goal of swapping out the 4-HT dependent intein for a cortisol dependent intein. We constructed the Kanamycin-4HT-Intein in the pET16b vector, and then transformed DH5-alpha. We picked colonies, grew liquid cultures, mini-prepped, and performed a diagonstic digest to confirm our insert was present. (See image of gel, well 3 shows undigested construct in pet16b, well 4 shows diagnostic digest confirming construct is present)
+
     <p> We tested the ability of the 4-hydroxytamoxifen (4-HT) dependent intein to splice in bacteria, rather than yeast in attempts to improve its characterization, and with the ultimate goal of swapping out the 4-HT dependent intein for a cortisol dependent intein. We constructed the Kanamycin-4HT-Intein in the pET16b vector, and then transformed DH5-alpha. We picked colonies, grew liquid cultures, mini-prepped, and performed a diagonstic digest to confirm our insert was present.  
−
<img src="XXX_Intein pic_XX" />
+
  
−
We then transformed our construct into BL-21 DE3 and induced expression with IPTG. To test intein splicing, we made agar plates containing either 1) Ampicillin, as a positive control, 2) Kanamycin, as a negative control 3) Kanamycin and 4-HT. We plated the BL21 DE3 and found that no bacterial colonies grew on the Kanamycin alone, or Kanamycin and 4-HT containing plates, but there was growth on the Ampicillin containing plates. If the intein was in fact splicing caused by 4-HT, we would expect to see growth on the Kanamycin and 4-HT containing plate. Therefore, this demonstrated that the intein construct was not splicing and Kanamycin Resistance was not being produced.
 
  
 +
<figure style="width:50%;margin-left:5%">
 +
<img src="https://static.igem.org/mediawiki/2018/0/05/T--Queens_Canada--intgel1.png"" alt='1ker'/>
 +
<figcaption>See image of Kanamycin-4HT-Intein diagnosstic digest gel, well 1 is a DNA Ladder, well 2 contains empty pET16-B, well 3 shows undigested construct in pet16b, and well 4 shows diagnostic digest confirming construct is present
 +
</figcaption>
 +
</figure>
 +
 +
 +
<figure style="width:50%;margin-right:5%">
 +
<img src="https://static.igem.org/mediawiki/2018/1/13/T--Queens_Canada--intgel2.png" width="600px" height="400px" alt='2ker'/>
 +
<figcaption>Wells 1,3,5,7,9 are undigested pET16b containing the Kanamycin-4HT-intein, and wells 2,4,6,8,10 are pET16b and intein digested with NotI.
 +
</figcaption>
 +
</figure>
 +
</p>
 +
<p>
 +
 +
We then transformed our construct into BL-21 DE3 and induced expression with IPTG. To test intein splicing, we made agar plates containing either 1) Ampicillin, as a positive control, 2) Kanamycin, as a negative control 3) Kanamycin and 4-HT. We plated the BL21 DE3 and found that no bacterial colonies grew on the Kanamycin alone, or Kanamycin and 4-HT containing plates, but there was growth on the Ampicillin containing plates. If the intein was in fact splicing caused by 4-HT, we would expect to see growth on the Kanamycin and 4-HT containing plate. Therefore, this demonstrated that the intein construct was not splicing and Kanamycin Resistance was not being produced.
 +
<br><br>
 
To discern why the intein was not splicing we ran a SDS-page on induced BL21-DE3 expression. We grew 2 liquid culture of BL21 DE3, induced expression of T7 RNA polymerase with IPTG, then incubated both for 4 hours at 37C. We then added 10μm 4-HT to one of the liquid cultures and incubated at 37C for an additional 1 hour to allow splicing of the intein to occur.
 
To discern why the intein was not splicing we ran a SDS-page on induced BL21-DE3 expression. We grew 2 liquid culture of BL21 DE3, induced expression of T7 RNA polymerase with IPTG, then incubated both for 4 hours at 37C. We then added 10μm 4-HT to one of the liquid cultures and incubated at 37C for an additional 1 hour to allow splicing of the intein to occur.
 
We then lysed the cells by addition of lysozyme and protease inhibitor containing lysis buffer, followed by sonication for 10 seconds. We then collected 1ml of the mixed fraction, and stored at -20C. We then centrifuged the remainder of the fraction at 8k RPM for 5 minutes, and collected 1ml of the soluble fraction. The remainder of the soluble fraction was tossed, and the insoluble fraction was resuspended and 1 ml was collected as the insoluble fraction.
 
We then lysed the cells by addition of lysozyme and protease inhibitor containing lysis buffer, followed by sonication for 10 seconds. We then collected 1ml of the mixed fraction, and stored at -20C. We then centrifuged the remainder of the fraction at 8k RPM for 5 minutes, and collected 1ml of the soluble fraction. The remainder of the soluble fraction was tossed, and the insoluble fraction was resuspended and 1 ml was collected as the insoluble fraction.
−
 
+
<br><br>
 
We then ran a SDS-page to determine protein expression, and if splicing had occured.
 
We then ran a SDS-page to determine protein expression, and if splicing had occured.
  
Line 46: Line 61:
 
</p>
 
</p>
  
−
 
+
<figure style="width:70%;margin-left:15%">
−
 
+
<img src="https://static.igem.org/mediawiki/2018/f/fb/T--Queens_Canada--sdsintein.png" width="600px" height="450px" alt='nolinker'/>
−
    <img src="XXX new part here XXXX" />
+
<figcaption> Wells 1,2,3 are incubated without 4-HT and the wells 4,5,6 are incubated with 4-HT. Wells 1 and 4 are the mixed fraction of the bacteria following lysis and sonication. Wells 2, 5 are the soluble fraction. Wells 3 and 6 contain the insoluble pellet. M: Mixed Fraction, S: Soluble Fraction, P: Pellet Fraction
 +
</figcaption>
 +
</figure>
 +
<br></p><p>
  
 
<h4>References</h4>
 
<h4>References</h4>

Revision as of 18:59, 15 October 2018

Improve

Parts

Previous Parts 4-HT Dependent Intein - http://parts.igem.org/Part:BBa_J32009

The 4-hydroxytamoxifen (4-HT) dependent intein splices in the presence of 4-HT in Yeast which allows for the ability to control the production of a desired engineered protein in a dose-dependent manner. [1] In the article published by Buskirk et al. (2004), the 4-HT dependent intein was tested and found to be effective in a variety of contexts, including kanamycin resistance, Green Fluorescent Protein, and Beta-galactosidase

Previous Parts 4-HT Dependent Intein - http://parts.igem.org/Part:BBa_K2612001

We tested the ability of the 4-hydroxytamoxifen (4-HT) dependent intein to splice in bacteria, rather than yeast in attempts to improve its characterization, and with the ultimate goal of swapping out the 4-HT dependent intein for a cortisol dependent intein. We constructed the Kanamycin-4HT-Intein in the pET16b vector, and then transformed DH5-alpha. We picked colonies, grew liquid cultures, mini-prepped, and performed a diagonstic digest to confirm our insert was present.

1ker
See image of Kanamycin-4HT-Intein diagnosstic digest gel, well 1 is a DNA Ladder, well 2 contains empty pET16-B, well 3 shows undigested construct in pet16b, and well 4 shows diagnostic digest confirming construct is present
2ker
Wells 1,3,5,7,9 are undigested pET16b containing the Kanamycin-4HT-intein, and wells 2,4,6,8,10 are pET16b and intein digested with NotI.

We then transformed our construct into BL-21 DE3 and induced expression with IPTG. To test intein splicing, we made agar plates containing either 1) Ampicillin, as a positive control, 2) Kanamycin, as a negative control 3) Kanamycin and 4-HT. We plated the BL21 DE3 and found that no bacterial colonies grew on the Kanamycin alone, or Kanamycin and 4-HT containing plates, but there was growth on the Ampicillin containing plates. If the intein was in fact splicing caused by 4-HT, we would expect to see growth on the Kanamycin and 4-HT containing plate. Therefore, this demonstrated that the intein construct was not splicing and Kanamycin Resistance was not being produced.

To discern why the intein was not splicing we ran a SDS-page on induced BL21-DE3 expression. We grew 2 liquid culture of BL21 DE3, induced expression of T7 RNA polymerase with IPTG, then incubated both for 4 hours at 37C. We then added 10μm 4-HT to one of the liquid cultures and incubated at 37C for an additional 1 hour to allow splicing of the intein to occur. We then lysed the cells by addition of lysozyme and protease inhibitor containing lysis buffer, followed by sonication for 10 seconds. We then collected 1ml of the mixed fraction, and stored at -20C. We then centrifuged the remainder of the fraction at 8k RPM for 5 minutes, and collected 1ml of the soluble fraction. The remainder of the soluble fraction was tossed, and the insoluble fraction was resuspended and 1 ml was collected as the insoluble fraction.

We then ran a SDS-page to determine protein expression, and if splicing had occured. We subsequently found one paper that had previously utilized dose-dependent intein splicing in bacteria, however this paper further tagged intein constructs with a maltose-binding protein.[2] Therefore, this further demonstrates the 4-HT dependent intein to be insoluble in Bacteria, unless its solubility is drastically increased through the use of solubility tags such as Maltose-binding protein.

nolinker
Wells 1,2,3 are incubated without 4-HT and the wells 4,5,6 are incubated with 4-HT. Wells 1 and 4 are the mixed fraction of the bacteria following lysis and sonication. Wells 2, 5 are the soluble fraction. Wells 3 and 6 contain the insoluble pellet. M: Mixed Fraction, S: Soluble Fraction, P: Pellet Fraction

References

  1. A. R. Buskirk, Y.-C. Ong, Z. J. Gartner, and D. R. Liu, Directed evolution of ligand dependence: small-molecule-activated protein splicing., Proc. Natl. Acad. Sci. U. S. A., vol. 101, no. 29, pp. 10505–10, Jul. 2004.
  2. Skretas G, Wood DW. Regulation of protein activity with small-molecule-controlled inteins. Protein Science : A Publication of the Protein Society. 2005;14(2):523-532. doi:10.1110/ps.04996905.