Team:ULaVerne Collab/Results

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Results: Part 2

The results explained on this page include the improvements discussed on our Results: Part 1 page.

PCR Gels

Figure 1.Gel electrophoresis ran at 110V for 30 minutes. Lane 1 is APEX 500 bp ladder, lane 2 is PCR purified LacP+RFP at the correct basepair length (924 bp), lane 3 is PCR purified proinsulin at the correct basepair length (1068 bp), NL 5.50 Single Chain Proinsulin at the correct basepair length (not used for this project; 1002 bp), and lane 4 is long lasting single chain insulin at the correct basepair length (1002 bp). This gel electrophoresis result supports that the LacP+RFP gene block was properly PCR'd. Therefore, the RFP expression observed in Figure 2 and Figure 3 can be attributed to the expression of this DNA sequence.

Interpretation: Based off of our gels we believe that our PCR products are the correct sizes because they were within the base pair regions that we were expecting to find them in. pSB1C3 had an expected length of 2070bp and was found around the 2000bp band on the ladder. RFP had an expected length of 924bp and was found between the 500bp band the 1000bp band on the ladder. Proinsulin had an expected length of 1068bp and was found around the 1000bp band on the ladder. Long Lasting Insulin had an expected length of 1002bp and was found around the 1000bp band on the ladder.

Transformations

Figure 2. Transformation of Proinsulin and Long Lasting Insulin into DH5-Alpha (E.coli) cells, shows colony growth. Figure 3. Transformation of Proinsulin + RFP Construct (BBa_K2958013)into BL21 cells glowing red due to successful RFP expression.

Interpretation: All four plates show Gibson Assembly Transformations into BL21 cells and successful growth of cell colonies. Although our colonies for the Proinsulin construct and the Long Lasting constructs did not express RFP, we decided to screen some colonies to check if we had a successful transformation.

Colony Screen

Figure 4. Colony PCR screening of Proinsulin and Long Lasting Gibson Assembly into BL21 cells using OneTaq Polymerase. Expected band size for Proinsulin is 1992bp and the expected band size for Long Lasting is 1926bp.

Interpretation: A successful colony PCR for Proinsulin includes both our proinsulin(1992bp) and RFP( 924bp)gene blocks together to form a band of around 1992bp. Similarly, a successful colony PCR for Long Lasting Insulin includes both our insulin(1002bp) and RFP( 924bp)gene blocks together to form a band of around 1926bp. Based off of our gel, our results signify that we were able to produce a band sized of around 1992bp for proinsulin in wells 2,4,5,6,7,8,9 and 1926bp for Long Lasting Insulin in wells 3,4,5,7,8,10.

Restriction Digest Using E and P Sites

Figure 5. Proinsulin Restriction Digest using EcoRI and PstI restriction sites compared to computer generated Digest using Serial Cloner.

Interpretation: Since we are using two restriction sites we should expect to see two bands. One Band will indicate our vector (pSB1C3) and the second band will indicate our proinsulin(1068bp) and long lasting (1926bp) gene blocks with RFP. Our Proinsulin does indeed show a band around 1068bp with a vector of 2070bp. The location of these bands also match closely to the computer generated Digest on serial cloner where the vector is around the 2000bp band and proinsulin is around the 1000bp band. Our long lasting insulin at first appears to not have worked because there is only one band, however, the two bands that are produced from using these restriction sites are very similar in base pair length. The first band would be our vector which is 2070bp, and the second band is our Long Lasting Insulin 1926bp. Since these bands are similar in size, they seem to create one large band. To check our work, we also ran this digest on the computer generated digest on Serial Cloner where we also got one band around the 2000bp band.

Restriction Digest Using Eco-RI-HF

Figure 6. Proinsulin Restriction Digest using Eco-RI HF restriction site compared to computer generated Digest using Serial Cloner.

Interpretation: Since we are only using one restriction site we should expect to see one band which will include all of our parts including vector (2070bp), RFP (924bp), and Insulin gene blocks(Proinsulin=1068 and Long Lasting= 1926bp). Our proinsulin digest should have one band around 4062bp and our long lasting should have a band of around 3996bp. As can be seen from our results, we do have bands in these areas which also match the computer model of the restriction digest which shows a band around the 4000bp band.

SDS-Page

Figure 7. Proinsulin SDS-Page

Interpretation: Protein gel of the RFP + Proinsulin construct, courtesy of iGEM Team_Moscow 2019. According to this protein gel, our construct appears to have worked as expected, as seen by the bands at the expected proinsulin fusion construct band and expected RFP band in the (+) lysate of the protein gel (indicated by the red arrow). The expected molecular mass of the proinsulin is 31.75 kDa, and the expected molecular mass of the RFP is 25.38 kDa. Although we see see the appropriate band sizes in this protein gel, we cannot confirm that this is our desired protein because it is from an overnight sample. If we wanted to confirm these bands as being a part of our construct then we would have to perform Nickel-NTA purification and still end up with the same bands at 31.75 kDa for Proinsulin and 25.38 kDA for RFP in our elution samples during the purification. Additionally we would have to perform Western Blot analysis, and ELISA assay to further characterize our parts.

Analysis Summary

From these results we can conclude that we had successful PCR, Gibson Assembly transformations, and Restriction Digests. We found that trying to purify our protein through Nickel-NTA purification proved very challenging. We believe the reason for this could be due to either lack of space between proteins for the nickel beads to bind to the HIS tag, or HIS tag being an ineffective method within itself. Unfortunately we were not able to get a protein gel to show that protein purification was successfully accomplished using Nickel beads. Due to the Nickel purification we were not able to isolate insulin using TEV protease, or characterize using Glucose uptake, or ELISA.

Future Plans

Some plans for the future would be to try and use another tag other than the HIS tag such as the HA tag or the GST tag. We would also need to find a cost effective method to purify the insulin such as using another metal bead, column chromatography, or aptamers. We were also advised to perform a Western Blot analysis when collaborating with the Biohacking crew at Open Insulin, however due to budget restrictions we were not able to afford this type of characterization.