Team:Sao Carlos-Brazil/Parts

Project Parts

BBa_K3273000 DelURA

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
Ura3 cassette promotes homologous recombination in Saccharomyces cerevisiae, it contains a homology region to the yeast ura3 gene, present in its genome, in chromosome V. This part was used in order to create an auxotrophic mark for screening. This screening method is inspired by the principle of operation of BBa_K2407301 part.

BBa_K3273001 Serratia marcescens nucA nuclease

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This part is a CDS that produces Serratia marcescens NucA nuclease. This enzyme is capable of cleaving both DNA and RNA, either single or double-stranded. It was implemented in our project as part of a kill switch in order to degrade the transgenic DNA and prevent it from being available for bacteria and other organisms in the environment.

BBa_K3273003 HXT6 promoter

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
The HXT6 promoter from Saccharomyces cerevisiae is a famous glucose repressible promoter. In nature it is part of the HXT6 gene that encodes a high-affinity glucose transporter. We used it to active our Kill switch in low glucose concentration.

BBa_K3273004 Hybrid promoter ADH2/TDH3 repressed by glucose

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
The hybrid promoter was developed to be repressed by glucose. The two upstream activator sequences (UAS) of the ADH2 promoter confer the repression characteristics at high glucose concentration. We fused -185 to -300 of the ADH2 promoter into the saccharomyces cerevisiae TDH3 promoter.

BBa_K3273006 AGA2 protein fused with melanin binding peptide 4D

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
The display protein AGA2 (BBa_K2027010) was fusioned with the melanin binding viral heptapeptide 4D (BBa_K2027010) by a flexible linker (GGGS)x4. The purpose of these constructions was to create a melanin armor on the surface of the yeast.

BBa_K3273007 Mammalian Bax

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This part is a CDS that produces a mammalian Bax protein, that has a pro-apoptotic function and can initiate apoptosis in mammals, yeast and even plant cells according to certain manuscripts. It was applied to the projects' kill-switch as one of the cell death mechanisms

BBa_K3273008 Composite ADH2/TDH3 nucA

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This part is composed of the BBa_K3273004 promoter, which is expressed in low glucose concentration, followed by a Kozak sequence BBa_J63003, then Serratia marcescens nucA nuclease BBa_K3273001 and the BBa_K2637017 terminator. It is a molecular kill switch activated in low glucose concentrations, that could prevent the DNA to be available to environmental organisms.

BBa_K3273009 Composite HXT6 Bax

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This composed part has the Hxt6 promoter BBa_K3273003, expressed in low glucose concentration, followed by the Kozak sequence BBa_J63003, Mammalian Bax apoptotic protein BBa_K3273007 and the BBa_K2637017 terminator. It is a kill-switch regulated by glucose concentration.

BBa_K3273010 Composite MET 25 nucA

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This composite part consists of the BBa_K165000 methionine induced promoter, followed by the Kozak sequence BBa_J63003, S. aureus nucA nuclease BBa_K1159105 and CYC1 terminator BBa_K2637017. It was designed to be used as a kill-switch system controlled by methionine concentration.

BBa_K3273011 Aga1

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This part is an Agglutinin, a yeast cell membrane protein. It was used in our project as a docking point for Aga protein and the rest of the genetic construction in order to create a display. It was based on BBa_M45091, with codons optimized for yeast.

BBa_K3273013 nucA S. aureus

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This nucA is an enzyme from Staphylococcus aureus that degrades DNA and RNA either single or double stranded. This part is based on BBa_K1159105, with codons optimized for yeast

BBa_K3273014 4D Melanin-Binding Heptapeptide

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
Repetitive 4D Melanin-Binding Heptapeptide. This sequence is based on BBa_K2027010, with codons optimized for yeast.

BBa_K3273015 Flexible linker 4x (GGGS)

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
Peptidic linker used to fuse proteins. This part was based on BBa_K1486003, with codons optimized for yeast, and size doubled.

BBa_K3273016 AGA2/linker/4D

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This composite part has the TDH3 promoter (BBa_K530008), followed by the Kozak sequence (BBa_J63003), then the display protein AGA2 (BBa_K416000) fused with the viral melanin binding heptapeptide 4D (BBa_K3273014) by a flexible linker (GGGS)x4 (BBa_K3273015) and the ADH1 terminator (BBa_J63002). The purpose of this construction was to create a melanin protection on the yeast surface, making it UV resistant.

BBa_K3273017 Composite TDH3/Kozak/Aga1/ADH1

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This is a composite part. It starts with the TDH3 promoter (BBa_K530008), followed by the Kozak sequence (BBa_J63003), then AGA1 cds (BBa_K3273011) and ADH1 terminator (BBa_K392003). It was used in our project as a docking point for Aga2 protein and the rest of the genetic construction in order to create a display

BBa_K3273018 Composite Promoter/RBS/Uracil-DNA glycosylase

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This is a composed part that produces the Uracil-DNA glycosylase of D. radiodurans. This enzyme is capable of repairing mutations caused by UV radiation exposure. It has a strong constitutive promoter and a strong RBS. It was designed to make E.coli UV resistant

Best Basic Part

Part:BBa_K3273001 Serratia marcescens nucA nuclease

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This part is a CDS that produces Serratia marcescens NucA nuclease. This enzyme is capable of cleaving both DNA and RNA, either single or double-stranded. It was implemented in our project as part of a kill-switch in order to degrade the transgenic DNA and prevent it from being available for bacteria and other organisms in the environment. It’s capability of degrading diverse types of genetic material makes it a very robust mechanism against transgenic genetic material propagation.

Best Composite Part

Part:BBa_K3273016 AGA2/linker/4D

Designed by: Bruno Batista, Fernando de Souza, Isabelle Taira, Lucas Boldrini
This composite part has the TDH3 promoter (BBa_K530008), followed by the Kozak sequence (BBa_J63003), then the display protein AGA2 (BBa_K416000) fused with the viral melanin binding heptapeptide 4D (BBa_K3273014) by a flexible linker (GGGS)x4 (BBa_K3273015) and the ADH1 terminator (BBa_J63002). The purpose of this construction was to create a melanin protection on the yeast surface, making it UV resistant. This part is the most important genetic construction in our project.

Best Parts Collection

Our part collection has two main sectors: the display and the kill switch. The display sector contains innovative parts that serve as a way of accumulating melanin on a yeast cell wall (and additionally repairing damaged DNA in prokaryotic organisms), in order to confer it resistance against ultraviolet radiation. Many parts are required to make it happen according to our design. The kill switch sector is composed of three different promoters (two are activated by small amounts of glucose and one by small amounts of methionine) and three different death mechanisms (two nucleases and one pro-apoptotic protein), promoting cell death in both molecular and cellular levels. Our collection is composed by the following parts:

  • BBa_K3273000
  • BBa_K3273001
  • BBa_K3273003
  • BBa_K3273004
  • BBa_K3273006
  • BBa_K3273007
  • BBa_K3273008
  • BBa_K3273009
  • BBa_K3273010
  • BBa_K3273013
  • BBa_K3273014
  • BBa_K3273015
  • BBa_K3273016
  • BBa_K3273018