Team:Austin UTexas/HP/Gold Integrated
Integrated Human Practices
Optimization of Golden Gate Assembly
1 - 5 Bridging Sequences
Many of the teams we talked to, including Texas Tech and Rice University, either did not use Golden Gate Assembly (GGA) or did not use it as their main technique for molecular cloning and were not intimately familiar with the process. Given our experience with GGA, we decided to make our kit simpler to use. We designed standardized bridging sequences that reduced the number of parts in a GGA reaction, increasing the likelihood of a successful assembly. Using this method, we were able to make multiple assemblies expressing different antibiotic resistance genes 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, containing multiple parts of interest, such as a promoter, 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 to the ends of the PCR products. These products are then made into parts plasmids, using BsmBI and T4 DNA ligase. The end product is a self-replicating plasmid that acts to store the part for use in a larger assembly. The BsaI sites on the insertion sequence are retained for future assemblies using multiple part plasmids.
Multiple part plasmids are combined in a Golden Gate Assembly reaction. Each part is digested with BsaI and ligated back together. However, the success of reaction, that is when all part types are successfully ligated together to create an entire plasmid, decreases as the number of parts increases. Therefore, larger, more sophisticated plasmids tend to be difficult to make.
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.
Better Selectable Markers
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.
Integration of Feedback from Synthetic Biology Experts
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.
In addressing Dr.Davies' suggestion that we show the kit can work in multiple organisms, we attempted to express a variety of our assemblies in non-model organisms. These organisms included:
- Serratia marcesscnes
- Serratia
- Staphylococcus epidermidis ATCC 35984
- Staphylococcus epidermidis ATCC 12228
- Psuedomonas chloritidismutans
- Vibrio nitragens
- Lactobacillus plantarum
Success was only confirmed in a few of these organisms. However, these experiments were informative in that they demonstrated the variety of transformation and growth procedures necessary to genetically manipulate non-model organisms and underscored the strengths and weaknesses of our kit.
See Demonstrate Page
1 Tube Reaction Feedback
Rice University
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.
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.
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.