Artificial sweeteners can avoid health hazards associated with added, purified sugars. However, certain artificial sweeteners are poorly tolerated. For example, aspartame must be avoided by individuals with phenylketonuria. Some people strongly dislike the flavors of artificial sweeteners. Thus, there is a demand for a variety of sugar alternatives both for health reasons and for personal preference.

Thaumatin is a protein present in the tropical plant Thaumatococcus danielli and is considered approximately 3000 times as sweet as sugar¹. Earlier research suggests that the sweetness of thaumatin can be modified by mutating individual amino acids involved with binding the human sweetness receptor protien². I propose cloning, expressing and purifying thaumatin for testing formulations of sugar substitutes. In future work, the sequence will be mutated and the changes in flavor and shelf life will be examined.

Kent, R. Sweet proteins – Potential replacement for artificial low calorie sweeteners. Nutr J. 2005;4: 5.

Thaumatin will be expressed in Pichia pastoris strain GS115, obtained from Invitrogen. The recombinant protein will be secreted into the growth media, and purified by nickel affinity chromatography. To accomplish this, the thaumatin open reading frame will be placed under control of the AOX1 promoter. This promoter is strongly induced by adding methanol to the growth media. After the protein is synthesis, its secretion will be driven by the native signal peptide of the thaumatin gene. A six histidine tag will be fused in frame to the C-terminus of the thaumitin coding sequence. This tag will be used in the affinity purification step using Ni-NTA chromatography. Transcription of the engineered mRNA will be terminated by an AOX1 terminator. The final designed sequence can be viewed at ​Final Construct .

Once transformed in P. pastoris, cells containing the plasmid will be selected for using the antibiotic zeocin. The plasmid provides resistance to zeocin via the BleoR gene.

Prior to transformation into the expression host, the plasmid will be assembled and transformed into E. coli 5-alpha cells obtained from New England Biolabs. E. coli containing the plasmid will be selected for with the antibiotic zeocin.

The gene will be cloned into the plasmid pPICZ A, whose original sequence is found here: ​pPICZ A . This sequence was imported using a file downloaded from SnapGene. The plasmid is distributed by Invitrogen. The plasmid provides the AOX1 promoter, the 6x-His tag, and the AOX1 terminator.

mRNA will be reverse transcribed using an oligo(dT) primer. This primer anneals to the poly(A) tail of eukaryotic mRNA.

The reverse transcription reaction is prepared as follows, according to the supplier's instructions for Superscript III, which can be viewed here (links can be added with ctrl-K or the link icon at the top of the editor window):

1 μL of 50 μM oligo(dT) primer

1 μL of Thaumatococcus danielli RNA at 100 ng/μL

1 μL of 10 mM dNTP mix

10 μL of water

This mixture is heated to 65°C for 5 minutes, then incubated on ice for 1 minute.

The following are added:

4 μL of 5X First-Strand Buffer

1 μL of 0.1 M DTT

1 μL of RNaseOUT Recombinant RNase Inhibitor

1 μL of SuperScript III Reverse Transcriptase

Mix and incubate at 50°C for 60 minutes.

Inactivate the reaction by heating at 70°C for 15 minutes.

The sample is now cDNA which can be used directly as a template for PCR. No purification is needed.

Thaumatin will be amplified using primers ​Thaumatin Forward and ​Thaumatin Reverse to yield ​Thaumatin I PCR product . Thaumatin forward has a melting temperature of 60.3°C to the matching cDNA sequence, calculated using the default parameters of Primer3. Thaumatin Reverse has a melting temperature of 60.8°C.

As suggested by the enzyme manufacturer, the annealing temperature will be T_m + 3°C, or 63°C.

Since the amplicon is less than 1 kb, a 15 second extension time is sufficient.

After amplification, the PCR product will be seperated on an agarose gel. I expect a single 728 bp band. The PCR reaction will be cleaned with a silica column [No other details about electrophoresis or purification are necessary]

As recommended using the double-digest finder tool (https://nebcloner.neb.com/#!/redigest), EcoRI and SalI can be used in a double digest using NEB buffer 3.1.

After the reactions are completed, the digested plasmid and PCR product are purified using a silica column.

Ligations are performed using T4 DNA ligase obtained from New England Biolabs.

The cloning reaction is used to transform chemically competent DH5-alpha cells obtained from New England Biolabs. The transformation protocol is derived from instructions found here.

Cells were plated on LB agar plates containing zeocin and grown overnight.

The pPICZ plasmid does not allow for blue-white colony selection, so individual colonies are screened for successful ligations using Sanger sequencing. Colonies are grown overnight in LB media containing zeocin.

Plasmids are purified from saturated media using a silica column [No other details needed here].

Plasmids are sequenced using ​Sequencing primer , which binds 39 base pairs upstream of the coding sequence [Many expression vectors are designed to use a T7 or M13 sequencing primer. You may use these if they are present in your vector, but make note of this in the write up and include a primer file in your project inventory].

A sequencing reaction of 900 bp read length will fully cover the thaumatin reading frame [If needed, design additional primers to sequence the your entire gene.]

Validated plasmids will be transformed into P. pastoris strain GS115 [Use a strain of E. coli instead] as follows and will be plated on YPD agar containing zeocin.

Enzymes, plasmids and strains used for this cloning project are indicated below.