Team:ULaVerne Collab/Failed-results
General Outline of Procedures
We began our project with one experimental construct and one positive control construct. To create this novel insulin we increased the isoelectric point from 5.4 of native insulin to 7.3. Modifications in the B chain obtained from Insulin Lispro for fast-acting insulin properties as its patent has expired (Zwebb, 2018), and the 12 a.a. linker chain from the Australian iGEM Team to link the A and B chain together. To increase the isoelectric point, the A chain is replaced with different a.a. at A18 (N18H) and A21 (G21D). To utilize fast-acting properties from Insulin Lispro, a.a. changes in the B chain were made at B28 (K28P) and B29 (P29K). We also implemented combinations of a Histidine tag, Ecotin tag, TEV tag, or R tag within the circuit.
K608014 Amplification:
Figure 1.
Amplification of rehydrated K608014 gene from iGEM plate kit. Well 1 contains the 1 kb ladder. Well 2 contains [1uL] K608014. Well 3 contains [5uL] K608014. Expected band size for both is 0.768kb and expected mass is 42ng.
PCR with overlap regions:
Figure 2.
Gel containing PCR parts and expected band sizes. Well 1 is 1 kb ladder. Well 2 is K608014 (0.768kb). Well 3 is Wild Type Insulin, (0.921kb). Well 4 is Experimental Insulin, (0.874kb). Well 5 is vector, (2.070kb).
Gibson Assembly Transformation:
Figure 3.
E. coli. strain DH5 alpha cells transformed with pSB1C3+ K608014+ wild type (Fig 4. A and B) and pSB1C3+ K608014+ experimental (Fig 4. C and D) using Gibson Assembly. Transformations were plated on LB Agar plates with CAM resistance and incubated at 37ºC overnight. Cell colonies expressing RFP are present.
Overnight Colonies
Figure 4.
Overnight colonies grown in LB media with CAM. Incubated overnight in a shaker at 37ºC at 225 rpm. Image 5A is control, and image 5B is from the experimental plates.
Colony PCR:
Figure 5.
Colony PCR Gel Electrophoresis. Expected band size for the wild type is 1.689kb and 1.642kb for the experimental. Wells 2-5 contain the wild type circuit and are around 1.5-2.0 kb. Wells 6-9 are experimental Gibson PCR. Well 6 is about < 1.0 kb and wells 7-9 are about 1.5-2.0 kb.
Protein Gel
Figure 6.
Protein gel: Well 1- Ladder, Well 2- (+) Ctrl. Overnight, Well 3- (-) Ctrl. Overnight, Well 4- (+) Ctrl. Cell lysis, Well 5- (-) Ctrl. Cell lysis, Well 6- (+) Ctrl. Sample and Nickel Beads, Well 7- (+) Ctrl. 1st wash, Well 8- (+) Ctrl. 1st Elution, Well 9-(+) Ctrl. 2nd Elution.
When combining all of our parts together (Ecotin + HIS + TEV) we should expect to see a protein around 27kDa. We see this expected protein size in our positive control, but then we seem to lose it when we perform cell lysis and nickel purification. This 27kDa band does not appear in the negative control (BL21cell), which led us to believe that this band could possibly be our protein of interest.
Analysis Summary: Based off of our gel results we believe that we have the correct DNA bands and have successfully gibson assembled our parts. We also believe that our transformation into DH5-alpha cells was successful because we were able to get red cell colonies due to the expression of RFP in our circuit. However, we seem to be losing our protein when we perform cell lysis and nickel purification.
Troubleshooting: Some alternative methods that we incorporated into our second set of constructs is use osmotic shock to extract our protein as well as cell lysis. By doing this we hope to get a band around 27kDa in our cell lysis wells. We also added a 6xGGS flexible linker to both sides of our HIS tag to allow it more space to bind to the nickel beads. We decided to make these modifications because we noticed that one reason we were losing our band may have been because our Ecotin tag and TEV tag were hindering our HIS tag from binding to the nickel beads due to their large size. We also decided to include an inducible promoter to control how much protein we are expressing which will be beneficial when we characterize.