Team:Penn/Synbio

Overview

After we had gleaned enough information about the microbiology of AMB-1, we began focus more on its applicability in synthetic biology. We began to explore the literature for transformation protocols. However, in our search, we found that very little transformation data or clear procedures existed for this strain of bacteria. During the course of our work with AMB-1, we consolidated various protocols and developed methods of our own in order to form a clear, optimized transformation protocol for future research teams to use.

Upon developing this “master-protocol”, we attempted transformation of the AMB-1 strain with the PYMB essentials BioBrick.

This vector contains an AMB-1 origin of replication, a replication gene and an AMB-1 specific promoter (pmsp1), on a PSB1C3 backbone. The backbone contains chloramphenicol resistance. If we were to successfully transform the vector into AMB-1 and grow it on chloramphenicol coated plates, this would indicate that the AMB-1 had received the shuttle vector and that the transformation was successful.

Eventually, after attempting to transform AMB-1, we found that our transformations with the shuttle vector PYMB essentials had key components missing from the sequence. We designed a new shuttle vector to transform AMB-1. We are introducing this plasmid to the iGEM community with the hope that it spurs future exploration of AMB-1 and that it inspires other teams to work with uncharacterized strains.

As we worked to transform AMB-1, we developed a more optimized transformation protocol. The protocol we developed is outlined here:provide link to the protocol

Our additions/findings to the original protocols are:

1. We outlined a protocol for the preparation of an aerobically grown cell culture of AMB-1 from an anaerobic culture. This is the first step of transformation. provide link to the protocol
2. We determined the optimal electroporation settings and buffer for transformation. provide link to materials/methods
3. The growth of bacteria on plates after recovery using the procedure outlined in the protocols found in literature says takes 2 weeks. We found that this technique can be optimized if one of two plating techniques outlined below are used to reduce oxygen exposure:
1. Plating the colonies on 1% agar directly, and then wrapping the plates in parafilm to reduce produces colonies in 20-24hours.
2. Plating cells and growing the cultures in an anaerobic chamber produces colonies in 10-12 hours.

PYMB Transformation Problems

As we proceeded in our attempts to transform AMB-1 with PYMB essentials, we found that despite our scrupulous adherence to the protocol, our transformation was unsuccessful.

We attempted to design an experiment that would address why AMB-1 was not growing on plates with antibiotic. We plated the bacteria at every step of transformation in order to address the success of each step. (Table 2.1)

From this experiment, we were able to determine that AMB-1 cells are viable are viable in aerobic cultures, after suspension in TES buffer, after electroporation, and after recovery. Therefore, in our next steps in troubleshooting transformation, we attempted to address the following two possibilities for why it was not successful.

We obtained the PYMB shuttle vector from a previous BioBrick that attempted to only retain key components of an original shuttle vector in an effort to reduce its length. The plasmid was constructed by an estimation of the length of the replication origin. Also, other parts of the plasmid expect for the replication gene were omitted. The original plasmid included pUC19 ligated on BamHI site with pMGT, an endogenous plasmid found in Magnetospirillum magneticum MGT-1. However, the BioBrick only retained the essentials of this large vector.