Team:Yale/notebook

Lessons from Week -1

Dan began his research for the summer two weeks before the other researchers arrived. This was the first week any iGEM researcher was in the lab, so it was focused on preparing the lab and assessing the viability of two of our possible projects. The project which was started this week focused on porting Multiplex Automated Genome Engineering (MAGE) from E. Coli, in which the technique is proven to work, to Cyanobacteria and Sinorhizobium bacteria.

In order to port MAGE from E. Coli to other organisms, we had to first find inducible promoters that worked in Cyanobacteria and Sinorhizobia, and then beta-homolog proteins (reference to MAGE section of our wiki) for both organisms. At this point in the project, we began with finding cyanobacterial inducible promoters. We found promoters in Cyanobacteria which should, in theory, be inducible, and one constitutive promoter. The promoters were a nitrate-inducible promoter nirA, an iron-inducible promoter isiAB, a green-light-inducible promoter pCpG2, and a constitutive promoter, psaA. We designed and ordered primers to isolate all of these promoters from their respective backbones, whether they were Synechococcus sp. 7002 or Synechocystis sp. 6803. We also focused our efforts on growing up copies of a backbone plasmid, BBa_K125000, which was to contain our primers.

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Lessons from Week 1

This week served as an orientation and planning period. The graduate mentors showed us around our lab space, and we practiced some lab techniques (such as proper aseptic technique and a transformation protocol in E. coli) with the Isaacs lab graduate students. The plan is to begin working with our rhizobia strains (Sinorhizobium meliloti 1021 and Rhizobium tropici CIAT) next week; we ordered some cyanobacterium Synechococcus sp. PCC 7002 from the Pasteur collection, but it is backordered and may take some weeks to arrive.

We laid out a series of experiments which would eventually allow us to express a nonspecific recombinase (beta-homolog) to incorporate foreign DNA into our organism's genome; this process is at the heart of the MAGE technique. Much of this week was spent identifying recombinases which could potentially function in our organisms through literature and BLAST searches. We also spent a significant amount of time identifying inducible promoters (either native to our organisms or within the BioBrick Registry) which we could use to express our recombinase.

The first step in our plan is to determine optimal growth conditions, transformation protocols, and selection screens for our strains. Once this is complete, we can test the effectiveness of our identified promoters by placing them upstream of a fluorescent reporter; we will use the yellow-fluorescent citrine protein since its emission wavelength does not overlap with the autofluorescence wavelengths of our cyanobacteria. After identifying the most effective promoter (lowest leakiness and highest expression level when induced), we can replace the citrine gene in our construct with our synthesized beta-homolog genes. Testing the effectiveness of our recombinases will be a bit of a challenge; we need an assay to be implemented in each organism that will allow us to quantify mutagenesis efficiency.

While all of this is going on, we will also need to knock out the mutS gene in each of our organisms. mutS is involved in identifying nucleotide mismatches during DNA replication. Since MAGE is founded upon such mismatches, it is in our best interest to allow them to go unnoticed by the cell's DNA proofreading systems. Silencing mutS should allow us to do this.

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Lessons from Week 2

For Rhizobium, Holly, Lionel, Jessica, and Erin tried to determine the optimal growth conditions. Following the example of literature, this team inoculated LB broth as well as different antibiotic mixtures of Tryptic Soy Broth and LB with the two strains of Rhizobium we had acquired from the Handelsman lab (Rhizobium tropici CIAT 899 and Rhizobium etli CE3). Rhizobium etli CE3 showed no growth, and, therefore, was dropped as a target for research. Rhizobium tropici CIAT 899 showed growth in all three mixtures of media; however, it showed the fastest growth in 50% Tryptic Soy Broth/ LB.

For Cyanobacteria, Dan inoculated both Synechocystis sp. 6803 and UTEX 2973 in BG-11. Along with this, Danny read literature and determined the best proteins to use for increasing the efficiency of MAGE in cyanobacteria. Colin searched for protocols that could be used for transformation, growth, and various other important processes.

For the lab overall, we attempted to electroporate various plasmids into E. Coli to improve our technique. However, these efforts were met with limited success.

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Lessons from Week 3

This week, we focused on obtaining and using new plasmids, pKT230 and pZE21G, because our prior plasmid, BBa_K125000 was causing various problems, from being improperly annotated online to stubbornly refusing to be isolated using a maxi prep. However, very little actual lab work was done this week. In regards to non-lab work, we researched and described a way to knock out the mutS gene in both Cyanobacteria and Rhizobia and explored the possibility of also porting a CRISPR/Cas9 system into Cyanobacteria or Rhizobia.

For lab work: we successfully miniprepped pKT230 and pZE21G out of E. Coli once the OD was 0.611. The Jacobs-Wagner lab at Yale also graciously gave us S. Meliloti cultures to use: #1021, #WM249, and #MB501. S. meliloti WM249 and MB501 are derivatives of Rm1021 with transposon insertions permitting the efficient electroporation of E. coli plasmid DNA. WM249 contains a Tn5-233 element encoding gentamicin resistance, whereas MB501 (obtained from M. Barnett, Stanford University) contains the same Tn5-233 swapped for trimethoprim resistance. We were also able to isolate the isiAB promoter from Cyanobacteria Synechococcus PCC 6803 using the PCR procedure outlined in the procedural notes. However, the ends of the promoter were custom-made to be gibson-assembly-ed into the BBa_K125000 plasmid, so if we end up switching plasmids we’d need to re-isolate the promoter.

Let me explain CRISPR. CRISPR (clustered regularly interspaced short palindromic repeats) is a recently discovered system from certain bacterial adaptive immunity systems that uses guide RNA and the Cas9 proteins to create gene knockouts and gene replacements. Guide RNA locates a specific homologous sequence in the DNA following a PAM sequence. The Cas9 protein then causes a double stranded break in the DNA (see the graphic below put together by the Wuhan University iGEM team in China). Currently, we have found in literature that Synechococcus does not have a fully functional CRISPR-Cas system. This means that imported systems will work more effectively due to lack of cross talk. We also found that cyanobacteria are capable of non-homologous end joining (NHEJ). This means that cyanobacteria are prime targets for the porting of CRISPR- Cas systems. In order to test the ability of the imported CRISPR-Cas systems, we will target the UreC gene, a gene which confers sensitivity to solutions of urea and nickel sulfate. Already, we have designed the plasmids for the guide RNA and the Cas9 protein. Cas9 will be placed on a low copy number plasmid (Bba_125000) with an inducible promoter such as isiAB or nirA. gRNA will be placed on high copy number plasmid (KT230) with a constitutive promoter such as psaA. If necessary, the cas9 protein can be placed next to a tetR promoter, a promoter found in E. Coli but not natively in cyanobacteria. In addition, recently it has been found that CRISPR works in Arabidopsis and several strains of rhizobium. However, no CRISPR has been found in the strains of Rhizobia we intend to use. It has also been shown that Rhizobia exhibits NHEJ. Whether or not pursuing CRISPR would be useful is an idea that needs to be discussed.

In order to port MAGE into Cyanobacteria and Sinorhizobia, the mutS gene must be knocked out. Why? MutS functions in the DNA mismatch repair pathway, which is a highly conserved system from prokaryotes to higher eukaryotes. The mutS protein recognizes a DNA mismatch, but since DNA mismatch is absolutely needed for the MAGE mechanism, a mutS knockout (KO) is essential for high mutagenesis efficiency. We decided that the best way to do this would be with a Flp-FRT Recombination. Here is the strategy for mutS Knockout in Synechococcus sp. PCC 7002:

1) Amplify and assemble KO construct: 1 kb Homology Arm (UpF) → FRT → kanR → FRT → 1 kb Homology Arm (DownF)

2) Transform KO construct into PCC 7002

3) Recombination event occurs between KO construct and cell genome

4) Screen for recombinants by culturing on kanamycin/PCR verify mutants

5) Transform plasmid containing Flp gene into cell; induce Flp expression

6) Flp cuts out kanR cassette, leaving an FRT site scar (Note: Flp-containing plasmid also contains another selectable marker gene, such as spcR)

7) Remove Flp-containing plasmid by heat shock

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Lessons from Week 4

This week, we made progress in amplifying cyanobacteria and rhizobium promoters. We performed multiple antibiotic resistance assays and transformation experiments in both types of bacteria as part of our attempt to create a framework for propagating and selecting nonmodel organisms.

All of the primers for our cyanobacteria promoters have arrived. Dan worked on amplifying them so that they can be assembled with citrine into one of the vectors we have and tested for strength. Unfortunately, due to a power outage that halted the PCR run, no amplifications were successful.

One of the bigger issues we handled this week was the cyanobacteria growth media. We ordered freshwater BG-11 from Sigma-Aldrich, but we needed marine BG-11 to grow PCC 7002. We researched and found the reagents necessary to prepare MN marine medium from a seawater base in our lab.

Our rhizobium transformation experiments gave us mixed results. Using electroporation, R. tropici was able to take up the KT230 plasmid, but S. meliloti 356, 370, and 371 demonstrated kan and spec resistance and we were not able to determine whether the transformation was successful for those strains. We also experimented with conjugation as an alternative transformation method. We found a protocol for conjugating E. coli to UTEX 2973 and began to incubate E. coli containing pKT230.2 When UTEX 2973 reaches OD750 0.5, we will proceed with conjugation.

We performed several rhizobium antibiotic resistance assays to determine whether our strains of rhizobium have natural resistance to certain antibiotics. The results suggested that none of our strains have kanamycin resistance, but R. tropici and S. meliloti 371 have spectinomycin resistance. We also performed a rifampicin assay to determine whether we could eventually use MAGE to induce resistance to rifampicin in rhizobium. The assay worked with 2x1010 cells, but even 20 uL/mL rifampicin was below the limit of detection (see Fig. 1). Our goal moving forward is to redo this assay at lower concentrations.

We also met with Professor Dellaporta, one of our PIs, about the possibility of using ligation-independent cloning (LIC) as an alternative to Gibson Assembly. With LIC, the BsaI T4 DNA polymerase generates long complimentary sticky ends between the vector and insert and eliminates the need for ligase. The advantage is that one LIC cloning vector can be used repeatedly to build many promoter and beta homolog constructs without PCR amplifying the large vector backbone each time. However, we would need to order new primers and spend a week constructing the LIC vector.

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