Team:Penn/Synbio

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.

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.

1) Plasmid Problems

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.

Due to this, we proposed that one reason for the failure of transformation is that this BioBrick is missing key components essential to its function that have been eliminated from the original sequence. It was possible that an essential part of the origin of replication removed was required for successful transformation of the gene.

Another concern is that the chloramphenicol resistance was part of the PSBIC3 backbone and did not have an AMB-1 specific promoter preceding it. The reduced expression due to the E.Coli promoter may not lead to resistance to chloramphenicol.

2) Error in the electroporation technique

We also wanted to ensure that the error was not in the electroporation itself, before we started to explore the possibility of a faulty plasmid. We designed an experiment to eliminate the possibility that a faulty electroporation machine or a flawed protocol was impacting our results.

In order to eliminate any doubt about electroporation, we chose to use the same protocol (substituting SOC broth for TES buffer) to transform E.Coli with the same plasmid. This is possible because the PYMB Essentials BioBrick is on the PSBIC3 backbone which has the E.Coli origin of replication and replication gene. Success would allow us to deduce that the problem is not in the protocol or our implementation of the protocol. We plated the E.Coli at every step of transformation keeping as many variables constant as possible. (Table 2.2)

The results of this experiment indicated to us that E.Coli cells are viable in aerobic cultures, after suspension in TES buffer, after electroporation, and after recovery. The electroporation was successful and the results imply that:

1. The device is effective
2. E. Coli rep gene is functional in PYMB
3. Chloramphenicol resistance is expressible in E. Coli
4. The protocol is feasible

The proposed next steps after this experiment were:

1. Redesign a shuttle vector BioBrick because the problem is most likely that the PYMB essentials plasmid is defective.

From this experiment, we were able to conclude that the problem with transformation was most likely due to a faulty shuttle vector.

Supporting literature

We also investigated the paper that isolated a native plasmid in Magnetospirillum magneticum MGT-1 and created a shuttle vector of AMB-1. This paper concluded that the shortest region required for replication in AMB-1 was more than 2 kB and included the AMB-1 origin of replication and rep gene. This region also included a long sequence of nucleotides that was essential for replication for the replication of the plasmid. In addition to this, the exact sequence of the origin of replication has not been isolated. The PYMB essentials biobrick only contains 80 nucleotides of what could be the origin of replication. This gives us more reason to believe that this part is not functional. []

Redesigning the Shuttle Vector

1. Chloramphenicol resistance doesn’t have AMB-1 promoter before it
2. Origin of replication is non-functional in PYMB

We worked to redesign the shuttle vector to eliminate these problems and create a working plasmid to successfully transform the rare strain of bacteria.

ori + rep + pmsp3

We began with a construction of the basic shuttle vector which contained the entire necessary sequences for transformation.

The plasmid illustrated above includes PSB1C3 from the stem loop on the left side going counterclockwise to the other stem loop on the right side.

The genes included in this in this plasmid are the origin of replication (ori) and the replication gene. We pieced the ori together in three pieces: the red 1.7 kb upstream piece, the green rbs + rep gene (already in the BioBrick Registry as BBa_K624003), and the 420 bp downstream piece.

After the ori and rep genes, the lime green piece is a promoter pmsp3 (the start site where the sequence of genes starts to be read) that is unique to our magnetic bacteria. It is a stronger promoter than the one included in the original PYMB essentials. People could “cut and paste” genes after this promoter to add them to the magnetic bacteria.

ori + rep + pmsp3 + chloramphenicol

Our second version of the shuttle vector has the chloramphenicol gene right after the AMB-1 specific pmsp3 promoter. As mentioned previously, this system eradicates the concern that the chloramphenicol gene is not being appropriately expressed because it is not near an AMB-1 promoter, but rather in the PSBIC3 backbone in the original construct.

Final shuttle vector

This final shuttle vector construct contains all of the pieces in the constructs described earlier, but provides a more concrete site for researchers to add their desired gene constructs. Counterclockwise to the 1.7 kb rep piece, there is now another pmsp3 promoter followed by a prefix, MCS (multiple cloning site), and suffix.

This final construct is therefore instrumental in the use of AMB-1 as a chassis. It allows for researchers to incorporate not only genes specific for the purpose of bioremediation, but also any other utilization of AMB-1 in research. Therefore, it is an important step in the characterization and development of this rare strain of bacteria in synthetic biology.