Difference between revisions of "Team:Georgia State/Design"
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<p>In May, our team discussed potential project ideas and concluded that we wanted to continue last year’s project, which was focused on developing an endotoxin detection system to replace the horseshoe crab blood-based Limulus amoebocyte lysate assay (LAL). The proposed detection system would function as follows: Factor C is activated by the presence of endotoxin, this triggers autocatalysis releasing the hCG to be detected by a pregnancy test strip. Our plan was to generate the Factor C- hCG fusion protein expression construct via overlap extension PCR. We performed numerous attempts to fuse four segments of Factor C cDNA as shown below. | <p>In May, our team discussed potential project ideas and concluded that we wanted to continue last year’s project, which was focused on developing an endotoxin detection system to replace the horseshoe crab blood-based Limulus amoebocyte lysate assay (LAL). The proposed detection system would function as follows: Factor C is activated by the presence of endotoxin, this triggers autocatalysis releasing the hCG to be detected by a pregnancy test strip. Our plan was to generate the Factor C- hCG fusion protein expression construct via overlap extension PCR. We performed numerous attempts to fuse four segments of Factor C cDNA as shown below. | ||
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<img src="https://static.igem.org/mediawiki/2018/3/30/T--Georgia_State--overlap_pcr.jpeg" width="50%"> | <img src="https://static.igem.org/mediawiki/2018/3/30/T--Georgia_State--overlap_pcr.jpeg" width="50%"> | ||
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After weeks of following different protocols and using different primers, they all resulted in incorrectly sized bands on the gels, we still had not succeeded in producing a full-length Factor C coding sequence, much less the desired Factor C-hCG fusion. By mid-July, we had begun to contemplate dropping factor C entirely. | After weeks of following different protocols and using different primers, they all resulted in incorrectly sized bands on the gels, we still had not succeeded in producing a full-length Factor C coding sequence, much less the desired Factor C-hCG fusion. By mid-July, we had begun to contemplate dropping factor C entirely. | ||
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<img src="https://static.igem.org/mediawiki/2018/d/d8/T--Georgia_State--pGEX_plasmid.jpeg"> | <img src="https://static.igem.org/mediawiki/2018/d/d8/T--Georgia_State--pGEX_plasmid.jpeg"> | ||
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Revision as of 01:07, 18 October 2018
Design
In May, our team discussed potential project ideas and concluded that we wanted to continue last year’s project, which was focused on developing an endotoxin detection system to replace the horseshoe crab blood-based Limulus amoebocyte lysate assay (LAL). The proposed detection system would function as follows: Factor C is activated by the presence of endotoxin, this triggers autocatalysis releasing the hCG to be detected by a pregnancy test strip. Our plan was to generate the Factor C- hCG fusion protein expression construct via overlap extension PCR. We performed numerous attempts to fuse four segments of Factor C cDNA as shown below.
After weeks of following different protocols and using different primers, they all resulted in incorrectly sized bands on the gels, we still had not succeeded in producing a full-length Factor C coding sequence, much less the desired Factor C-hCG fusion. By mid-July, we had begun to contemplate dropping factor C entirely.
During this time, we also traveled to Baltimore, Maryland to participate in the 2018 Mid-Atlantic meet up. After presenting our project idea at the time, we received feedback and advice that helped shape our new project idea. We found that we were most excited about hCG as a detection mechanism as opposed to factor C, and that combined with our previous trouble making the fusion protein led us to drop factor C from our project entirely. From this point we aimed to produce a functional hCG protein fused to glutathione S transferase (GST), a protein widely used in fusion constructs to aid in purification and detection of desired synthetic proteins.
We set out to ligate our hCG coding part into the pGEX expression vector so that we could express the hCG protein as a fusion with GST and test it on a pregnancy strip to make sure that hCG would work as a signal in a fusion protein.
We successfully inserted our hCG coding part into the pGEX vector and set about expressing and purifying the hCG-GST fusion protein At the beginning of August, we realized that we were still having a hard time getting our hCG-GST protein to function properly. Although we were seeing bands on our Western blots,when we tested our protein samples with test strips we got no reaction. The test strips gave a clear positive reaction when exposed to even tiny concentrations of commercially available hCG, but nothing with our purified samples. We decided to do additional research concerning producing an hCG-GST protein and found possible complications.
We found that hCG expressed in E. coli can form inclusion bodies that get stuck in the membrane and may impede proper purification and activity. Although we had been trying to synthesize the full beta chain of hCG, we read that smaller segments of the chain were sufficient to activate the hCG receptor. Perhaps small segments could also stimulate a positive signal on a pregnancy test strip while avoiding the problem of inclusion body formation. We decided to try producing a small segment of the beta chain, the beta 3 loop, which had been shown to bind to and activate the hCG receptor.
We designed a new coding part to express the hCG beta 3 loop. As before we wanted to express it as a fusion protein with GST to test its suitability as a signal in a fusion protein detector system. We successfully ligated the part into pGEX, proceeded with a protein expression and purification via glutathione column, and examined the results by SDS-PAGE with coomassie staining and Western blot probed with anti-hCG.