Difference between revisions of "Part:BBa K2974500"
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
−
<partinfo>BBa_K2974500 short</partinfo>
+
<partinfo>BBa_K2974700 short</partinfo>
  
−
BBa_J23106 Toehold GFP is a construct developed by the 2019 Lambert iGEM team in order to be utilized as a biosensor. This part is designed to be used in conjunction with BBa_K2550001. From previous experience, the team was aware that a strong promoter such as BBa_J23100 from the Anderson Series of Promoters may produce overexpression or unintended transcription and expression of the reporter gene in a toehold construct. In order to improve upon these side effects, the team replaced the promoter from BBa_J23100 to BBa_J23106. In addition, the team replaced the LacZ reporter with eGFP to replace the color-based quantification system with a fluorescence-based quantification system.  
+
T7 Toehold LacZ is a construct that was developed to be applied as a biosensor. The part BBa_I732005 was submitted by 2007 UTSC iGEM that singularly included the LacZ gene encoding the Beta-galactosidase protein. In an effort to build on this biobrick and implement LacZ blue color expression as a biosensor, Lambert iGEM obtained a LacZ toehold switch construct assembled with a medium-strength T7 promoter BBa_J23106. The medium-strength promoter replaced the strong T7 promoter BBa_J23100 obtained from the Styczynski Lab at the Georgia Institute of Technology used in the 2018 Lambert iGEM project. The strong promoter was responsible for leakiness in the toehold, and the team sought to diminish this overexpression to improve the 2018 part BBa_K2550000 contributed by Lambert iGEM.  When assembled with a distinct RNA sequence complementary to the trigger sequence, the produced blue pigment expression can be characterized based on a Red, Green, Blue (RGB) scale.
 +
 
 +
 
 +
<strong>Description</strong>
 +
<br>
 +
Toehold switches are synthetic RNA strands that mimic messenger RNA sequences. They contain a complementary recognition sequence for a specific sequence of RNA stimuli , and a ribosomal binding site where a ribosome binds to initiate the translation of a reporter protein. The switch has a hairpin loop structure that is formed through binding to complementary sections of its own sequence. When in the presence of the complementary trigger sequence the hairpin loop opens to allow downstream expression. The Ribosomal Binding Site and starting sequence are concealed in the toehold switch until initiated. Switches can provide rapid, convenient, in-field detection that can be developed in both cellular systems and cell-free tests.
 +
<br>
 +
In the T7 LacZ Toehold construct, the lac operon is induced by lactose and IPTG (isopropyl β-D-1-thiogalactopyranoside); this region of the genome is responsible for transporting and metabolizing lactose. Within the lac operon, the gene, LacZ, codes for the B-galactosidase protein. When this protein is expressed, it breaks down X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) into galactose and an insoluble blue pigment. Therefore, when the gene is synthesized in the chassis, the colonies will appear blue. This mechanism was implemented in the toehold switch construct to identify the presence of specific substances.
  
−
<strong>Descriptions</strong>
 
  
 
<strong>Results</strong>
 
<strong>Results</strong>
 +
<br>
 +
Currently, the team is undertaking cloning workflow using Gibson Assembly to assemble these parts. The team used Polymerase Chain Reaction with Gibson primers of the designed LacZ fragments to amplify specific strains. The team then ligated these fragments and transformed using Dh5a cells, which grew colonies.
 +
<br>
 +
<br>
 +
<html>
 +
<body>
 +
<center><img src="https://2019.igem.org/wiki/images/8/85/T--Lambert_GA--GelElectrophoresis1.png" height="150px"></center>
 +
</body>
 +
</html>
 +
<br>
 +
<center><i>Figure 1. PCR products of LacZ fragments (approximately 3000 base pairs), wells 4, 5, and 7 show faint bands at around 3000 base pairs when referenced to the 2-log ladder in well 6.</i></center>
 +
<br>
 +
<br>
 +
<html>
 +
<body>
 +
<center><img src="https://2019.igem.org/wiki/images/1/12/T--Lambert_GA--OLacZ1.png" height="300px"></center>
 +
</body>
 +
</html>
 +
<br>
 +
<center><i>Figure 2. Transformation of the LacZ Toehold with the strong promoter, currently without pigmentation.</i></center>
 +
<br>
 +
<br>
 +
<html>
 +
<body>
 +
<center><img src="https://2019.igem.org/wiki/images/a/a2/T--Lambert_GA--NLacZ1.png" height="300px"></center>
 +
</body>
 +
</html>
 +
<br>
 +
<center><i>Figure 3. Transformation of the LacZ Toehold with the medium-strength promoter, currently without pigmentation.</i></center>
 +
  
−
The Medium Promoter Toehold GFP Biobrick (BBa_K2974500) was assembled into pSB3C5, a medium copy plasmid. After obtaining the BBa_J23106 promoter Toehold GFP gene block, the team digested the toehold insert using EcoRI-HF and PstI-HF and then confirmed the digest using gel electrophoresis. Furthermore, the team ligated the insert into a pSB3C5 vector digest and transformed the ligation with DH5α E.coli competent cells. The team then purified the DNA and sent it to sequencing, which came back successful. Sequencing results along with an additional gel confirmed the successful assembly of the toehold insert.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 17:27, 21 October 2019


Strong Promoter (BBa_J23100) Toehold GFP

T7 Toehold LacZ is a construct that was developed to be applied as a biosensor. The part BBa_I732005 was submitted by 2007 UTSC iGEM that singularly included the LacZ gene encoding the Beta-galactosidase protein. In an effort to build on this biobrick and implement LacZ blue color expression as a biosensor, Lambert iGEM obtained a LacZ toehold switch construct assembled with a medium-strength T7 promoter BBa_J23106. The medium-strength promoter replaced the strong T7 promoter BBa_J23100 obtained from the Styczynski Lab at the Georgia Institute of Technology used in the 2018 Lambert iGEM project. The strong promoter was responsible for leakiness in the toehold, and the team sought to diminish this overexpression to improve the 2018 part BBa_K2550000 contributed by Lambert iGEM. When assembled with a distinct RNA sequence complementary to the trigger sequence, the produced blue pigment expression can be characterized based on a Red, Green, Blue (RGB) scale.


Description
Toehold switches are synthetic RNA strands that mimic messenger RNA sequences. They contain a complementary recognition sequence for a specific sequence of RNA stimuli , and a ribosomal binding site where a ribosome binds to initiate the translation of a reporter protein. The switch has a hairpin loop structure that is formed through binding to complementary sections of its own sequence. When in the presence of the complementary trigger sequence the hairpin loop opens to allow downstream expression. The Ribosomal Binding Site and starting sequence are concealed in the toehold switch until initiated. Switches can provide rapid, convenient, in-field detection that can be developed in both cellular systems and cell-free tests.
In the T7 LacZ Toehold construct, the lac operon is induced by lactose and IPTG (isopropyl β-D-1-thiogalactopyranoside); this region of the genome is responsible for transporting and metabolizing lactose. Within the lac operon, the gene, LacZ, codes for the B-galactosidase protein. When this protein is expressed, it breaks down X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) into galactose and an insoluble blue pigment. Therefore, when the gene is synthesized in the chassis, the colonies will appear blue. This mechanism was implemented in the toehold switch construct to identify the presence of specific substances.


Results
Currently, the team is undertaking cloning workflow using Gibson Assembly to assemble these parts. The team used Polymerase Chain Reaction with Gibson primers of the designed LacZ fragments to amplify specific strains. The team then ligated these fragments and transformed using Dh5a cells, which grew colonies.


Figure 1. PCR products of LacZ fragments (approximately 3000 base pairs), wells 4, 5, and 7 show faint bands at around 3000 base pairs when referenced to the 2-log ladder in well 6.




Figure 2. Transformation of the LacZ Toehold with the strong promoter, currently without pigmentation.




Figure 3. Transformation of the LacZ Toehold with the medium-strength promoter, currently without pigmentation.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]