Team:Austin UTexas/HP/Gold Integrated

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Integrated Human Practices

Optimization of Golden Gate Assembly

Many of the teams we talked to, including Texas Tech and Rice University, either did not use Golden Gate assembly or did not us it as their main technique for molecular cloning and were not intimately familiar with the process. To make our kit simpler to use, we designed standardized bridging sequences that reduced the number of parts in a reaction and increased the likelihood of a successful assembly. Using this method, we were able to make many assemblies expressing different antibiotic resistances quickly.

Golden Gate Assembly is a molecular cloning method that uses modular, interchangeable parts. Parts are categorized into types according to their function. In order to make a full assembly plasmid that contains multiple parts of interest, such as a promoter of choice connected to a specific coding sequence and a specific origin, each part must first be put into a standardized entry vector. This entry vector is pYTK001, a GFP-dropout with a colE one origin of replication and a chloramphenicol resistance gene. Parts are cloned from a template plasmid using primers that add BsmBI and BsaI restriction sites. They are then made into transcriptional units by a first stage Golden Gate reaction that digests both the insertion sequence and the vector with BsmBI and ligates them back together. The end product is a self-replicating plasmid that acts to store the part for use in a larger assembly. The BsaI one sites on the insertion sequence are retained. This first stage transcriptional unit is termed a part plasmid

Multiple part plasmids are combined in a Golden Gate Assembly reaction. Each is digested with BsaI and ligated back together. However, the number of successful reactions, that is all part types ligating together to create the desired assembly, decreases as the number of parts increases. Therefore, larger, more sophisticated plasmids tend to be difficult to make.

Fig 1. Schematic of reaction to make a 1-5 Bridge. The 1-5 Bridge reduces the number of parts in a BsaI Golden Gate assembly reaction to make a full plasmid that contains all part types.

Our solution was to create a standardized bridging sequence that contained part types 1 through 5 to make the number of overall BsaI containing parts in the final reaction smaller. Instead of desgning primer that contain both BsaI and BsmBI sites on both ends, the 1-5 bridge only contains a BsaI sequence on the Type 1 Forward primer and on the Type 5 reverse primer. All other parts in the bridge contain only BsmBI sites. Therefore, when the parts are combined in a BsBI Golden Gate reaction, a single part that can be used in BsaI assembly is formed.

Optimization of Chromoproteins

Many of our chromoproteins, such as Red chromoprotein and E2-Crimson, express weakly or take days to portray the desired phenotype. To make them more useful selectable markers, we coupled the sequence to a strong, constitutive promoter. This created brighter colors that expressed quicker

When attempting to make assemblies, our experiments were often delayed when we attempted to use certain fluorescent proteins or chromoproteins because they either did not express, expressed weakly, or took many days to become visbile.

To solve this problem, we extracted a strong promoter, CP25, and a red chromoprotein from pSL1, with primers that added BsmBI and BsaI restriction sites. We then inserted that sequence into pYTK001 to make a part plasmid. Currently, we are attempting to make assemblies with this part.

Figure 2: Red chromoprotein coupled to a strong promoter inserted into an entry vector and expressed in E. coli.

Transformation of Assembly Plasmids into Mu Free Donors

Dr. Brian Renda of Gingko Bioworks emphasized that our kit would be limited by the ability of the plasmids to be inserted into the bacteria of interest as many can not be transformed with standard protocols. Therefore, we transformed the assembly plasmids into a strain of E. coli that can act as a plasmid donor in conjugations.

See Demonstrate Page

Rice University Collaboration

After discussing our kit with members of the Rice University iGEM Team, we designed methods to simplify Golden Gate Assembly reactions, redesigned our presentation of the kit, and amended our protocol for potential users.

1 Tube Reaction Feedback

Members of our iGEM Team gave the Rice University iGEM Team our one tube reaction to test in their lab. One reaction contained all our confirmed full assemblies and the other contain our assemblies for a single antibiotic, kanamycin. After performing the one tube test reaction, they sent us the pictures found in Figure 2.

Figure 3: These are the results of Rice's one tube electroporation with E. coli.

They also gave us feedback that we incorporated into our protocol and experimental design. They mentioned that it would be helpful to have the expected colony phenotype for each assembly in the protocol. Instead of the one tube reaction, they would have preferred that each assembly was sent in a separate tube which they believe would have been more amenable to the heat shock protocol they used.

While the E2-Crimson, shuttle vector assemblies did not initially express when our team performed the one tube reaction, Rice saw blue colonies on their plates.