Difference between revisions of "Team:Austin UTexas/Improve"

 
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<h3>BBa_xxxxxxx</h3>
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<h3>BBa_K2657004: RCP Phytobrick with a Strong Promoter</h3>
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<p>This Phytobrick is an improved version of the Biobrick BBa_K864100, which is the coding sequence for super yellow fluorescent protein (sYFP2). We added overall usability and stability to this sequence by not only turning it into level 0 Phytobrick for use in Golden Gate Assembly, but we removed mutational hotspot from the sequence, making it more stable.<p>
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<p>For this improved part, we modified BBa_E1010  and added the synthetic broad host range promoter, CP25, and an RBS sequence to our improved part. The broad host range promoter will be compatible with a higher number of bacterial strains. We also made the sequence functional in MoClo Assembly with BsaI by inserting the sequence into the PhytoBrick universal acceptor, a modified pSB1C3 backbone.</p>
 
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<p>Originally, BBa_K864100 had the IS10 sequence: 5' - gctnagc - 3', which resulted in an insertion in the region of: 5’ -acaggcgtagtaccg- 3’. In BBa_xxxxxxx (the improved part), the IS10 sequence has been removed and the area of the insertion sequence is now 5’ -actggggttgttcca- 3’, which reduces the palindromic nature of the sequence. Palindromes in the DNA cause potential hairpins, which prevents RNA polymerase from translating the DNA correctly. We also turned this part into a level 0 Phytobrick by assembling it with the Universal Acceptor, BBa_P10500. This in turn will allow researchers to use this very fluorescent protein gene in Golden Gate Assembly reactions using the enzyme BsaI.<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/c/ca/T--Austin_UTexas--KimRCPCP25_2.png">
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<figcaption><b>Figure 1.</b> The black circle encloses the successful transformation of the RCP phytobrick BBa_K2657004. The strong red color is clearly visible.</figcaption>
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<h3>BBa_K2657003: RCP Phytobrick</h3>
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<p>In order to improve a part already in the Biobrick Registry, we chose BBa_E1010, which expresses the red chromoprotein. We then designed primers that added BsmBI and BsaI restriction sites to the sequence and inserted it into PhytoBrick universal acceptor BBa_P10500 via BsmBI assembly. This created a Phytobrick that functions in Golden Gate Assembly (GGA) reactions.</p>
 
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<h3>BBa_rcppartp</h3>
 
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<p>We took BBa_E1010 and added restriction sites then inserted it into PhytoBrick universal acceptor, BBa_P10500, in order to create a phytobrick for use in Golden Gate Assemblies with Bsa1.</p>
 
 
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<img src="https://static.igem.org/mediawiki/2018/3/38/T--Austin_UTexas--RCPlevel0.PNG">
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<h3>BBa_K2657005: sYFP2 with Mutational Hotspot Removed</h3>
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<h3>BBa_linked</h3>
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<p>BBa_K2657005 is an improved version of the Biobrick BBa_K864100, which is the coding sequence for super yellow fluorescent protein (sYFP2). We increased the evolutionary stability of the sequence by removing a mutational hotspot. Originally, BBa_K864100 had an IS10 insertion sequence site, which resulted in an insertion in the region of: 5’ -acaggcgtagtaccg- 3’. In BBa_K2657005 (the improved part), the IS10 sequence has been removed and the area of the insertion sequence is now 5’ -actggggttgttcca- 3’. This sequence maintains the codon identity of the original sequence.</p>
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<p>For this improvement, we took BBa_E1010 (RCP) and added the synthetic, high copy number promoter, CP25, and an RBS sequence to our Phytobrick. This will increase the expression of the RCP. We also made the sequence golden gate compatible by inserting the sequence into the PhytoBrick Universal Acceptor BBa_P10500, for future use in Golden Gate Assemblies. By linking a Promoter/RBS with a coding sequence, this will increase the efficiency of the BsaI reaction in order to make transcriptional units in Golden Gate Assemblies.</p>
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<p>We then designed primers that added BsmBI and BsaI restriction sites to the sequence and inserted it into PhytoBrick universal acceptor, BBa_P10500, via BsmBI assembly. This created a Phytobrick that functions in Golden Gate Assembly reactions, which is useful due to its strong fluorescent character.</p>
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<img src="https://static.igem.org/mediawiki/2018/1/1f/T--Austin_UTexas--cp25plusRCP.PNG">
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<img class="center" src="https://static.igem.org/mediawiki/2018/5/5c/T--Austin_UTexas--KimSYFP2.png">
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<figcaption><b>Figure 2.</b> Colonies are non- fluorescent under blue light. White-circled colonies highlight what expected colonies look like with the PhytoBrick.</figcaption>
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Latest revision as of 02:44, 18 October 2018


Improve



BBa_K2657004: RCP Phytobrick with a Strong Promoter

For this improved part, we modified BBa_E1010 and added the synthetic broad host range promoter, CP25, and an RBS sequence to our improved part. The broad host range promoter will be compatible with a higher number of bacterial strains. We also made the sequence functional in MoClo Assembly with BsaI by inserting the sequence into the PhytoBrick universal acceptor, a modified pSB1C3 backbone.


Figure 1. The black circle encloses the successful transformation of the RCP phytobrick BBa_K2657004. The strong red color is clearly visible.


BBa_K2657003: RCP Phytobrick

In order to improve a part already in the Biobrick Registry, we chose BBa_E1010, which expresses the red chromoprotein. We then designed primers that added BsmBI and BsaI restriction sites to the sequence and inserted it into PhytoBrick universal acceptor BBa_P10500 via BsmBI assembly. This created a Phytobrick that functions in Golden Gate Assembly (GGA) reactions.



BBa_K2657005: sYFP2 with Mutational Hotspot Removed

BBa_K2657005 is an improved version of the Biobrick BBa_K864100, which is the coding sequence for super yellow fluorescent protein (sYFP2). We increased the evolutionary stability of the sequence by removing a mutational hotspot. Originally, BBa_K864100 had an IS10 insertion sequence site, which resulted in an insertion in the region of: 5’ -acaggcgtagtaccg- 3’. In BBa_K2657005 (the improved part), the IS10 sequence has been removed and the area of the insertion sequence is now 5’ -actggggttgttcca- 3’. This sequence maintains the codon identity of the original sequence.


We then designed primers that added BsmBI and BsaI restriction sites to the sequence and inserted it into PhytoBrick universal acceptor, BBa_P10500, via BsmBI assembly. This created a Phytobrick that functions in Golden Gate Assembly reactions, which is useful due to its strong fluorescent character.

Figure 2. Colonies are non- fluorescent under blue light. White-circled colonies highlight what expected colonies look like with the PhytoBrick.