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E.Cargo overview

IN SUMMARY

• We tried to create a modular Tat protein transduction domain Biobrick.
• We fused this to transcription factors in RFC25 format. This would allow us to transiently deliver proteins to a cell and thus test components of our LRC.

Rationale and Background

OUTLINE

Trans-Activator of Transcription (Tat) is an HIV gene that functions to increase the transcription level of viral genes. The Tat Protein Transduction domain (Tat-PTD) is an 11-amino acid peptide that allows the protein to traverse cell and nuclear membranes in order to localize to the nucleus.

As a result of years of study, it is now known that there are multiple methods by which this domain operates. Most commonly, the Tat-PTD interacts with heparin sulfate proteogycans (HSPGs) to cause lipid-raft mediated endocytosis. In the endocytosed vesicles, heparinase causes degradation of the heparin sulfate, which releases the bound Tat-PTD (and anything attached to it) into the cytoplasm¹

• Project was initially developed as a way of delivering scFv (single-chain antibody) to mammalian cells to fluorescently label cell structures.
• As the LRC project developed, we realized Tat-PTD could be used to test components of our circuit individually without the need to perform individual gene transformations
• To this end, we decided to test whether we could fuse Tat to transcription factors, and use these "Tat-TFs" to "remotely" induce gene expression.

Experimental Design

CONSTRUCTING TAT-TF

The Tat sequence was initially ordered with an anti-histone scFv attached by a polyglycine linker. Our intention was to express this Tat-scFv fusion, purify, label with rhodamine, and apply to mammalian cells. We would've expected fluorscence to localize to nuclear histones to confirm that Tat was carrying the antibody through a live cell membrane. Our DNA construct was synthesized by DNA 2.0.

• For our Tat-TF, we needed to put the Tat-ptd with RFC25 ends (optimized for protein fusion). We used PCR to attach sites to the existing circular Tat DNA, avoiding the process of resynthesizing. This was biobricked as our Tat-linker.

• The next step was to adapt the transcription factor for use with Tat. We decided to use two TFs from the registry, [http://partsregistry.org/Part:BBa_I732100 LacI] and AraC. (PART #s?) We made several modifications through PCR: (removal of LVA rapid degradation tag, adding 6-his tag for nickel-column purification, RFC25 sites)

RFC25_AraC_FW (basic/stacking calc Tm=55/55c) 5- TA GAATTC GCGGCCGC T TCTAGA TG GCCGGC atggctgaagcgcaaaatgatcc-3

Actual 3’ sequence desired (REV primer = reverse complement of actual): 5-CTGCAAACGACGAAAACTACGCT CATCATCATCATCATCAT TAATAA ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG-3

RFC25_AraC_Rev (basic/stacking calc Tm=55/55c) 5-TA CTGCAG CGGCCGC T ACTAGT ATTA ACCGGT TTATTA ATGATGATGATGATGATG AGCGTAGTTTTCGTCGTTTGCAG-3

RFC25_LacI_FW (basic/stacking calc Tm=57/53c) 5-TA GAATTC GCGGCCGC T TCTAGA TG GCCGGC atgaaaccagtaacgttatacgatgtcg-3

Actual 3’ sequence desired (REV primer = reverse complement of actual): 5- CGACTGGAAAGCGGGCAG CATCATCATCATCATCAT TAATAA ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG-3

RFC25_LacI_Rev (basic/stacking calc Tm=55/54c) 5-TA CTGCAG CGGCCGC T ACTAGT ATTA ACCGGT TTATTA ATGATGATGATGATGATG CTGCCCGCTTTCCAGTCG-3

• The linear PCR products of Tat-linker and transcription factors were then subcloned into pGEM-T Easy
• Perform BioBrick assembly to join Tat-linker to transcription factor in pGEM-T Easy
• This entire Tat-TF construct was double digested and ligated into pBlueScriptSK, an expression vector. We insert into competent XL1 Blues E. coli and use blue/white screening (Protocol here add link) and ampicillin resistance to select for successful transformants.
• We cut the transcription factor out of pGEM-T Easy, run out on a gel to confirm successful cut, extract bands, then insert alongside the Tat-linker in pBlueScriptSK using sites for protein fusion in RFC25. (SHOW STEPWISE DIAGRAM OF CONSTRUCTION)
• Transform our Tat-TF in pBlueScriptSK into BL21 cell line, using Amp+ selection.
• Grow overnight of successful transformants, harvest cells and use Ni-affinity column to isolate purified Tat-TF protein.

TESTING THE TAT-TF

• To test for efficacy of Tat-TF control, we decided to use two reporter constructs. The LacI reporter construct was taken straight from the registry. The AraC reporter construct was assembled from registry parts:
  

1. Production of CyanFP is controlled by pLacI, which is constitutively on and repressible by addition of LacI protein.

• [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0020 Cyan Fluorescent Protein]
• [http://partsregistry.org/Part:BBa_K091110 LacI repressible promoter]

2. CherryFP production is linked to pAraC, which is inducible by addition of the AraC protein.

• [http://partsregistry.org/Part:BBa_R0080 AraC inducible promoter]
• [http://partsregistry.org/wiki/index.php/Part:BBa_J06702 RBS-Cherry Fluorescent Protein-terter]

• We then transformed each reporter construct into E.Coli (XL1B) and froze them into glycerol stocks for future use.

Results/Future Direction

• Due to the gradual evolution of this project and the time constraints of our summer, we were unable to secure experimental data on fluorescence after assembling most of our constructs in the lab. That being said, we did plan a series of experiments to determine the efficiency of our Tat-TF delivery method.

• Our first step is to confirm our reporter constructs work as expected. We created a construct that allowed cells to constitutively produce one of our transcription factors, LacI (BBa_K324003). When we transform our LacI reporter cell line with this construct, we expect LacI to be produced on site and endogenously repress cyan FP production. Quantitative analysis using a fluorometric plate reader in our research facility would measure any changes in fluorescence relative to background levels. This would be repeated with AraC to verify function of our inducible reporter.

• The next stage of our project is to test Tat-TF's ability to translocate across the cell membrane. We plan to regrow the cell lines containing our reporter constructs, apply purified Tat-TF, and measure any changes fluorescence. We expect to see a decrease in cyan fluorescence in our repressible LacI reporter, and an increase in cherry fluorescence in our inducible AraC reporter. This would indicate that our cells are responding to the remote delivery of a transcription factor.

• Finally, we will prove that the Tat domain is responsible for membrane translocation by lysing some of our cells that are constitutively producing LacI and AraC. We will apply the lysate, containing our transcription factor, to our reporter cell lines and look for changes to fluorescence. Lack of induction/repression would show that ordinary transcription factors lack the ability to freely enter the cell nucleus. This would suggest that our Tat domain functions as expected.

References

1) Chauhan A, Tikoo A, Kapur AK, Singh M. The taming of the cell penetrating domain of the HIV Tat: myths and realities. J Control Release. 2007;117:148–62.

2) Wadia JS, Dowdy SF. Protein transduction technology. Curr Opin Biotechnol. 2002;13:52–56. doi: 10.1016/S0958-1669(02)00284-7.