Difference between revisions of "Team:Montpellier/WetLab ASA"

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<h3>Western Blot</h3>
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<p>Since we did not succeed to transform <i>Lactobacillus jensenii</i> properly, we studied the production and secretion of our antibodies in <i>E. coli</i> with the plasmid psb1C3 (because it is a high copy number plasmid). Negative control is <i>E. coli</i> without plasmid.</p>
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<p>The expected antibody sizes are the following:</p>
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<ul>
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<li>LaM-4 (with the signal peptide YncM) : 570 nt | 20,9 kDa</li>
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<li>LaM-4 (with the signal peptide CbsA) : 527 nt | 19,25 kDa</li>
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<li>LaM-4 (without the signal peptide) : 432 nt | 15,84 kDa</li>
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</ul><br/>
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<ul>
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<li>YLP20 (with the signal peptide YncM) : 855 nt | 31,3 kDa</li>
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<li>YLP20 (with the signal peptide CbsA): 813 nt | 29,81 kDa</li>
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<li>YLP20 (without the signal peptide) : 717 nt | 26,29 kDa</li>
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</ul>
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<img class="image_figure" src="https://static.igem.org/mediawiki/2018/5/54/T--Montpellier--wb1_mtp.png"/>
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<img class="image_figure" src="https://static.igem.org/mediawiki/2018/8/8f/T--Montpellier--wb2_mtp.png"/>
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<figcaption><span class="underline">Figure 7:</span> Western Blot results.</figcaption>
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<p>We can only see the presence of a protein with E-tag in the pellet, and a slight presence in the supernatant. So, we cannot said that there is a secretion but a least there is a production inside the cell.</p>
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<p>Moreover, the sizes observed do not correspond to our expectations, mainly for YLP20 which seems to small. It could be supposed that the protein has been cleaved. Currently, we can just confirm that our promoter works in <i>E. coli</i>.</p>
  
  

Revision as of 21:34, 17 October 2018

Antibodies

Designs

The following figure (Figure 1) is the caption for the next 3 figures of our designs. Orange frames are the variable parts between the constructs.


Figure 1 : Meaning of the icons for the 3 following figures.

We decided to design several constructions to produce YLP20, our favorite candidate with four differents signal peptides : CbsA, Epr, YncM and Yjfa (Figure 2). We chose RpsU promoter [1] that is a strong promoter specific to L. jensenii.
We tested different signal peptides that we introduced you before without forgetting the amino sequence “APVT” to avoid truncating the protein. Finally, we added the sequence that encodes YLP20 scFv and E-tag.

Figure 2 : Constructs that produce and secrete YLP20 with 4 different signal peptides.
Figure 3 : Constructs that produce and secrete LaM-4 (a control) with 4 different signal peptides.

To check the scFv production and conformation we designed constructions with LaM-4 instead of YLP20 sequence (Figure 3).
Indeed LaM-4 is a control [2]. It is an antibody against RFP & mcherry. This control allow us to check the efficiency of the promoter and the signal peptide.
The construction is very similar that the one with YLP20. The difference is just that we replaced the YLP20 sequence with the LaM-4 sequence.

Moreover, we would like to check signal peptides that we chose. Indeed, it is really important to verify the efficiency of the SP and determine new SP that are working in L. jensenii. We decided to fusion the SP to RFP to display the efficiency of the SP (Figure 4). For that, we used a plate reader and a flow cytometer.

Figure 4 : Constructs that produce and secrete RFP with 4 different signal peptides.

Experimental design to assess Antisperm Antibodies production and secretion

To verify if our constructs are working correctly (i.e production and secretion in the supernatant) we designed several experiments (Figure 5).

The first experiment consists of a Western-Blot (WB) to verify that the antibody is produced by the bacteria. Our scFv is fused to an E-tag, allowing for straigthforward detection using a commercial antibody.

The second experiment consists of an ELISA test using YLP12 (biotinylated as follow : YLPVGGLRRIGG-Lys(Biotin)) which is the target antigen for the YLP20 scFv. The idea is to use streptavidin plates to fix YLP12, so that he repulsion between biotin and streptavidin forces the peptides not to lie on the bottom of the plate and facilitate the binding antigen-antibody. Different ELISA tests would need to be performed : First, using a supernatant from a bacterial culture to see if YLP20 is produced and secreted. In parallel, an ELISA using the cell lysate would be useful to see if YLP20 is produced but not secreted.

Figure 5 : Experimental design to assess production and secretion of antisperm scFvs in Lactobacillus jensenii. All the constructions are cloned by Gibson Assembly in E. coli. After the constructions are sequence verified, L. jensenii is transformed by electroporation. To verify if both production and secretion of scFv are effective, a Western Blot would be performed. After that, an ELISA test run to see if the produced scFv ca link to the specific peptide YLP12.

Expected results

Figure 6 describes the possible outcomes of the Western Blot experiment. If everything in the YLP20 sequence is working, we will detect the scFv in both supernatant and pellet (cell lysate). If only the signal peptide is not working, we will have a signal only in the cell lysate. Finally, if the scFv is not well expressed, (e.g. because of the promoter, the RBS, or aggregation) we will have detect no signal neither from the supernatant nor from the pellet. As a positive control, we must run a WB on a strain containing a protein fused to an E-tag that we know is well expressed.

Figure 6 : Analysis of the WB results depending of what works and what does not work in the sequence.

Results

All the constructions were cloned in E. coli DH5ɑ. Currently, we did not succeed to transform L. jensenii with different antibody constructions.

Cloning

Part Designed Cloned Characterized
Antibody Signal Peptide
YLP20 CbsA YES YES YES
YncM YES YES YES
YjFA YES NO NO
EPR YES NO NO
LaM-4 CbsA YES YES YES
YncM YES YES YES
YjFA YES NO NO
EPR YES NO NO
RFP CbsA YES YES NO
YncM YES YES NO
YjFA YES NO NO
EPR YES NO NO
LaG-16–G4S–LaG-2 CbsA YES NO NO
YncM YES NO NO
YjFA YES NO NO
EPR YES NO NO
Western Blot

Since we did not succeed to transform Lactobacillus jensenii properly, we studied the production and secretion of our antibodies in E. coli with the plasmid psb1C3 (because it is a high copy number plasmid). Negative control is E. coli without plasmid.

The expected antibody sizes are the following:

  • LaM-4 (with the signal peptide YncM) : 570 nt | 20,9 kDa
  • LaM-4 (with the signal peptide CbsA) : 527 nt | 19,25 kDa
  • LaM-4 (without the signal peptide) : 432 nt | 15,84 kDa

  • YLP20 (with the signal peptide YncM) : 855 nt | 31,3 kDa
  • YLP20 (with the signal peptide CbsA): 813 nt | 29,81 kDa
  • YLP20 (without the signal peptide) : 717 nt | 26,29 kDa
Figure 7: Western Blot results.

We can only see the presence of a protein with E-tag in the pellet, and a slight presence in the supernatant. So, we cannot said that there is a secretion but a least there is a production inside the cell.

Moreover, the sizes observed do not correspond to our expectations, mainly for YLP20 which seems to small. It could be supposed that the protein has been cleaved. Currently, we can just confirm that our promoter works in E. coli.

References
[1] Xiaowen Liu, Laurel A. Lagenaur, David A. Simpson, Kirsten P. Essenmacher, Courtney L. Frazier-Parker, Yang Liu, Daniel Tsai, Srinivas S. Rao, Dean H. Hamer, Thomas P. Parks, Peter P. Lee and Qiang Xu. (2006). Engineered Vaginal Lactobacillus Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N. Antimicrob Agents Chemother Vol. 50, No. 10, p. 3250–3259
[2] Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., ... & Rout, M. P. (2014). A robust pipeline for rapid production of versatile nanobody repertoires. Nature methods, 11(12), 1253.


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