Team:Austin UTexas/Parts

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Parts

Overall, we submitted five Phytobricks

Basic Parts

BBa_K2657001: Tetracycline Resistance Gene

When observing the parts registry, we found that none of tetracycline resistance genes were Phytobrick compatible. Using primers, we added proper MoClo standard overhangs to each part and provided it with proper restriction sites — BsaI and BsmBI sites. The PCR products containing these overhangs were incorporated into the Universal Acceptor backbone BBa_P10500, which is a modified pSB1C3 backbone. Link to Part Page

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Figure 1: The transformants present on this plate do not have natural tetracycline resistances. Their growth on the Tet+LB plate indicates that they have received tetracycline resistance from the TetR part.

BBa_K2657002: RP4 Origin of Transfer

At the same time, while looking over the parts registry, we found no origin of transfer with Phytobrick overhangs. Using the same techniques that were used to make the TetR Phytobrick, we created the RP4 Phytobrick, which is a level 0 OriT Phytobrick. Link to Part Page. Here is also an example of the RP4 part used in conjugation: Link

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Figure 2: Conjugation of Serratia marcescens. The plates on the left show the red bacteria under visible light while the plates on the right demonstrate the organism's expression of GFP when viewed under blue light.

Improved Parts

BBa_K2657003: Red Chromoprotein Phytobrick

In order to improve a part already in the Biobrick Regisry, we chose the Red Chromoprotein-expressing, BBa_E1010. 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. Link to Part Page

BBa_K2657004: Red Chromoprotein with Strong Promoter cp25

For this improvement, we took BBa_E1010 (RCP) and added the strong synthetic constitutive promoter CP25, which functions in a broad range of organisms. This will increase the expression of the RCP. We also made the sequence Golden Gate Assembly (GGA) compatible by inserting the sequence into the PhytoBrick Universal Acceptor, BBa_P10500. By linking a Promoter/RBS with a coding sequence, the transformation efficiency of GGA reactions will increase since the number of parts in the assembly will be reduced. This will also allow BBa_E1010 to be a more useful selective marker since, in its current form, it only expresses weakly and takes one or two days to express at all. Link to Part Page

Figure 3: The black circle indicates the streaked transformants’ strong red color.

BBa_K2657005: Improved sYFP2 Phytobrick

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 the mutational hotspot. Originally, BBa_K864100, contained a palindromic sequence. Palindromes in the DNA cause potential hairpins, which prevents RNA polymerase from translating the DNA correctly. 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. Link to Part Page