Team:Shenzhen SFLS/Results

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Results

The aim of our experiments is mainly to confirm the expression of our protein. As described in lab protocol, all sample from induction and his-tag purification is sds-paged and we can quickly distinguish between the his-tagged target protein and other irrelevant ones. Therefore, we observe the protein and marker strips on the sds-gel to assess our outcomes.

The results of plasmid PCR

We conducted Plasmid PCR to confirm the validity of our protein sequence. The sequence is as follows:

• Grid 1: DNA marker

• Grid 2: mfp3

• Grid 3: Tyr

• Grid 4: mgfp5-masp1

• Grid 5: mgfp5

Fig 0: The UV image of DNA gel

The correct result of this DNA electrophoresis should produce a travelling distance of mfp3< mgfp5< mgfp5-masp1< tyr. Unfortunately, the grid which contains mgfp5-masp1 is broken due to unknown reasons and the electrophoresis fails to record the strips of DNA. However, the strips of other three proteins corresponds to their own molecular weight and shows the travelling distance relationship of mfp3< mgfp 5

The results of sds-page analysis

Note:

For mgfp5, we uses Prestained Color Protein Molecular Weight Marker (270kD to 6.5kD) from Beyotime. For more information about the marker and the scaling, visit https://www.beyotime.com/product/P0071.htm.

For tyr, we uses Prestained Color Protein Molecular Weight Marker (170kD to 10kD) from Beyotime. For more information about the marker and the scaling, visit https://www.beyotime.com/product/P0075FT.htm

(A)The results of mgfp5 characterization

At first, the sds-page images fail to record any strips in protein elution.

Fig 2: A picture showing no strips at all.

However, after our integrated human practice, we found out the reason behind failure to be the formation of inclusion body, which blocks the his-tag. In order to destroy the inclusion body and release the his-tag, we need to denaturate the protein to change its structure. With the consideration in mind, we repeated the experiment for another two times with denatured induction product and successfully capture strips on the sds-gel from the elution of his-tag purification.

Note: Due to the denaturation process, the first ten grids show messy strips that cannot be analyzed.

Fig 3: Denatured protein samples and wash buffer content

Grid one: Marker; Grid two: Cl from cell lysis; Grid three: Cl from supernatant; Grid four: Ft (sample run through tube); Grid five: Wash 1; Grid six: Wash 2; Grid seven: Wash 3; Grid eight: Wash 4; Grid nine: Wash 5

Fig 4: Elution to Fig 1 samples

Grid 1: Elution 1; Grid 2: Elution 2; Grid 3: Elution 3; Grid 4: Elution 4; Grid 5: Elution 5; Grid 6: Elution 6. Grids 7-10 are intentionally left blank.

Fig5: Fig 3 scanning image

Fig 6: Fig 4 scanning image

As shown in the purification figure (Fig 4), we observe strips of a bit above 6.5kD marker strip, which corresponds to the molecular weight of our mgfp5 protein (258 bp = 86 amino acids = 8.6 kD). Therefore, we can confirm that we have successfully produced the mgfp5 protein.

(B)The results of Tyr Characterization:

We conducted a few trails in effort to express the enzyme tyrosinase. The first two experiments, owing to the issue of inclusion bodies, failed to record any strips on the gel. After preparing for a third time. We successfully derive strips from the elution of his-tag purification.

(Note: We didn’t keep the failure record so only success data will be included. Also, as denatured protein Cl and washings will show messy strips during electrophoresis, we discarded Samples from Cl to W3 and keep only E1-E5)

Fig 7: The elution of Tyr

Grid 1: Elution 1; Grid 2: Elution 2; Grid 3: Elution 3; Grid 4: Elution4; Grid 5: Elution 5.

The results for mfp3:

Owing to time limit, we could only conduct one experiment on mfp3 and we only managed to finish the induction process. However, we have conducted DNA gel analysis on mfp3 to confirm the senquence length of our DNA. (As shown in the first picture).

The results of mgfp5-masp1:

Two experiments were conducted on the mgfp5-masp1 part to characterize it. We haven’t derived visible strips to confirm the production of this part due to time limit. However, we confirmed the sequence length using the PCR automatic analysis. (As shown in the first picture).