Part:BBa_K2455003

Cell-Penetrating USER Cassette

This biobrick contains an AsiSI/Nb.BSMI USER Cassette with a N-terminal nona-arginine (R9) Cell-penetrating peptide which gives proteins inserted into the cassette the ability to pass through plasma membranes. In addition the USER Cassette carries a N-terminal hexa-histidine tag which allows for easy purification.

For our study we demonstrated that proteins inserted into the cassette can be purified, using the histidine-tag, and subsequently transferred into E. coli cells through the use of the R9 peptide.

In addition; previous studies have shown proteins associated with R9, either covalently or non-covalently, to be able to enter a variety of cell types (Chang et al.).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Cell-Penetrating Peptides (CPPs) are small peptides, typically rich in basic residues, which are able to facilitate transport of a wide variety of cargoes across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain (Eudes & Chugh, 2008). In recent years research have been done into making synthetic CPPs, especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells (Chang et al.).

USER Cassette

The biobrick carries the AsiSI/Nb.BsmI USER Cassette which facilitates gene insertion through the use of USER cloning.

Figure 1: Insertion of genes using USER cloning. Gene constructs consisting of multiple pieces can be inserted through the creation of a series of pieces with matching overhangs. One such use could be to insert point mutations into the gene of choice. The outer most overhangs must match the USER Cassette for the insertion to work.

For more information on USER cloning see [http://www.cbs.dtu.dk/services/AMUSER/ Amuser] or [http://www.cbs.dtu.dk/services/AMUSER/instructions.php Amuser instructions].

Insertion Requirements

For insertion of genes into the USER Cassette genes have to be amplified with the primers carrying the two following overhangs:

Forward primer: CGTGCGAUCx

Reverse primer: CACGCGAUxx

Where U = Uracil and x = arbitrary nucleotide; these are needed to keep the inserted gene in-frame. AsiSI and Nb.BsmI have to be used to facilitate opening of the cassette.

Gene Insertion

In order to test the ability of the biobrick to facilitate protein import in E. coli we choose to insert two different fluorescent proteins into it, SYFP2 and mTag BFP.

Opening of USER Cassette

To facilitate insertion we first had to prepare the vector, this was done in a two step digestion reaction. First to open the cassette 42 µL purified vector was mixed with 2.5 µL AsiSI, 10 µL 10x Tango Buffer, and 45.5 µL Nuclease-free water. The mixture was then incubated for 3 hours at 37 degrees Celsius. 5 µL linearised vector was run for 20 min at 100 V on a 1 % Agarose gel to check for proper linearisation.

Figure 2: Linearization of modified pET102 vector carrying the Cell-Penetrating USER Cassette biobrick. Lane 1: undigested vector, Lane 2: AsiSI digested vector. Vector has a size of 6kbp.

Following digestion the vector was subjected to column-purified using [http://omegabiotek.com/store/product/e-z-n-a-gel-extraction-kit/ E.Z.N.A.® Gel Extraction Kit] and [http://omegabiotek.com/store/wp-content/uploads/2013/05/D2500.D2501-Gel-Extraction-Kit-Combo-Online.pdf standard protocol] for gel-extraction with 150 uL binding buffer being added in the first step.

Finally the vector was nicked by mixing the complete 50 uL elute from the column-purification with 1 uL Nb.BsmI, 6 uL 10x NEBuffer 3.1, and 3 uL Nuclease-free water. The mixture was incubated for 3 hours at 65 degrees Celcius.

Gene Amplification

In order to facilitate insertion of the SYFP2 and mTag BFP biobricks into the USER Cassette, the two genes were amplified using the following sets of primers in order to add the appropriate overhangs:

SYFP2:

Forward primer: CGTGCGAUCA-ATGGTTAGCAAGGGCGAAG

Reverse primer: CACGCGAUGA-TTTATACAGCTCATCCATACCCAGGG

mTag BFP:

Forward primer: CGTGCGAUCA-ATGAGCGAACTGATCAAAGAG

Reverse primer: CACGCGAUGA-ATTCAGTTTATGACCCAGC

And gel-extracted using [http://omegabiotek.com/store/product/e-z-n-a-gel-extraction-kit/ E.Z.N.A.® Gel Extraction Kit] and [http://omegabiotek.com/store/wp-content/uploads/2013/05/D2500.D2501-Gel-Extraction-Kit-Combo-Online.pdf standard protocol].

Insertion of BFP and YFP

Insertion of the gel-extracted PCR products was done by mixing 1 µL PCR product with 2 µL linearised vector, 0.5 µL CutSmart® Buffer, 0.5 µL USER® Enzyme, and 1 µL MQ H2O. The mixture was incubated in a PCR machine for 25 min at 37 degrees Celcius, 20 min at 25 degrees Celcius, 10 min at 20 degrees Celcius, and 10 min at 15 degrees Celcius.

Correct insertion was validated through PCR amplification of inserts and subsequent sequencing.

Purification and SDS-PAGE

Following correct insertion of the SYFP2 and mTag BFP biobricks into the Cell-Penetrating USER Cassette, expression was done in Escherichia coli BL21 under control of the T7 promoter.

Figure 1: THIS IS A PLACEHOLDER HOLDER HOLDER HOLDER...

Purification

Cells were harvested, re-suspended in a lysis buffer containing 25 mM Imidazole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.9), and 1.0 mM PMSF, and lysed by sonication. Cell debris was removed by centrifugation at low speed, and the supernatant was transferred to a fresh falcon tube and mixed with 100 µL Ni-NTA Agarose. The mixture was incubated for one hour on a cold room turning table, where after the beads were spun down and the supernatant removed. A series of elution buffers (0.5 M NaCl, 20 mM Tris-HCl; pH 7.9) with increasing Imidazole concentration was used to elute the bound proteins by consecutively adding 100 µL elution buffer, centrifuging the sample at low speed, and removing 100 µL eluate.

NOTE: Samples were kept on ice at all times and centrifugation took place at 4 degrees Celsius.

SDS-PAGE

In order to analyse the purity of the proteins,