A Successful Molecular Cloning: Overcoming Stubborn Structures

If you've followed our methods section, you'll be happy to know that we completed some important experiments on our path to making algal insulin a reality. First off, the proinsulin gene was cloned into the pASapI backbone this month, and experimentally verified using restriction digest, polymerase chain reaction (PCR), and DNA sequencing.

 

Fig. 1: Molecular Cloning of Proinsulin Coding DNA Sequence into the pASapI Chloroplast Expression Vector. (Completed April 2016)

 

Fig. 2: Top: The restriction digest of the predicted pASapI-proinsulin plasmid cut with HindIII and Xhol, with a predicted size of ~300bp if proinsulin gene was present. Left to Right: 100bp ladder, pASapI-proinsulin cut product (colony 1), pASapI-proinsulin cut product (colony 2), empty PASapI vector (control). Bottom: The simulated gel was run on SnapGene and also matched experimental results with the correct band sizes and location.

 

Fig. 3: A gel showing a positive PCR product with primers complementary to proinsulin sequence for 7/8 E. coli colonies sampled after transformation with the pASAPI-proinsulin vector. From left to right: Lane 1: 100bp ladder, Lanes 2-9: positive proinsulin PCR product (expected size, ~300bp), Lane 10: 100bp ladder, Lane 11: positive control on the far right.

 

Fig. 4: Sequencing results of the molecular cloning. The recombinant plasmid was sent for sequencing and the resultant reads were aligned to the plasmid map. All reads overlap the proinsulin coding sequence, with some minor point mutations in the open reading frame (pictured below)

 

Fig 5: Several point mutations were discovered in a small TTTT region in the coding sequence of the proinsulin gene. One colony with a G mutation that changes Phe to Tyr and another that changes Tyr to Phe, but there are two Phe amino acids immediately before hand so it should not be a major issue. We will continue to monitor the effect of this mutation on the gene.

Secondly, the pASapI-aaDa plasmid (inferring spectinomycin resistance) also worked in E. coli, indicating that the plasmid was ligated successfully.

 

Fig. 6: Testing of the spectinomycin resistance in E. coli (left, pASapI-aaDa, right, negative control, pASapI). Growth was observed in the presence of the antibiotic.

Wrapping up the first half of the experiment

Though we had run into some initial setbacks due to structural issues with the gene we had ordered (the ligating ends of the synthesized gene were misbehaving), we finally had a success which now paves the way for ultimately characterizing the expression of our gene in microalgae. What seemed to have fixed the ligation was using a 24 hour cutting time and a 36 hour ligation period and some very fresh, competent BL21 Turbo cells. Once we purify the proinsulin produced from the recombinant E. coli colonies and verify its presence with an immunoblot, all that will remain is verifying the expression in microalgae as well.

 

Fig. 7: The histidine purification columns are on the way!

We will use the polyhistidine purification tag on the recombinant proinsulin protein to isolate it from the cells we grow and then probe the solution with our anti-proinsulin immunoblot.

A great thank you!

Lastly, we greatly appreciate the patience and understanding of our supporters, especially when we had to start troubleshooting. Good science takes time. We are looking forward to completing the last few experiments very soon, and hopefully in conjunction with other crowd-funded scientific projects like the Open Insulin project, we can continue working to improve the human condition by making vital medicines more accessible and affordable for everyone.