Team:Minnesota/Judging

From 2010.igem.org

Judging Criteria

This section will include all the work we have done to satisfy competition requirements beyond our project.

Parts Submitted to Registry

Below is our list of parts that have been submitted to the Parts Registry.

Judges, we would like to draw your attention to the part EutSK. In this composite part, we have stacked all the 5 Eut bacterial microcompartment genes from Salmonella LT2. As demonstrated in our Results section, the recombinant Eut proteins from this part are expressed well in E.coli. We have also shown that co-expression of EutSK with a signal sequence-tagged reporter leads to localization of the reporter to a distinct point in the cell, suggesting that this part is able to form a functional shell which encloses the reporter protein.

Charecterization of mutated Lac Promoter

Cloning of the ethanolamine utilization microcompartment proteins relied upon the use of a vector created in the lab of our faculty mentor Dr. Claudia Schmidt-Dannert (Johnson et al, manuscript in prep). The plasmid contains a constitutively active mutant version of the E. coli Lac promoter: it has the RNA polymerase binding region, but lacks the repressor protein binding site (Schmidt-Dannert, 2000). The modified lac promoter has strong constitutive activity, and is a good candidate for consideration by other Registry users. To make our modified lac promoter more attractive (and useful) to other teams, we have characterized it using the flow cytometry technique known as Fluorescence Assisted Cell Sorting (FACS). To assay our promoter, we used 2 different E.coli strains - JM109 and DH5 alpha-pro- the latter constitutively expresses the Lac repressor protein (LacI). FACS was used to assay promoter activity in these 2 E. coli strains under varied levels of Isopropyl β-D-1-thiogalactopyranoside (IPTG), a compound known to inactivate the LacI protein. Our rationale was that as our modified lac promoter is constitutively active, neither the presence of LacI nor addition of IPTG should have any effect on the promoter output (GFP expression). The FACS data, presented in Figure 1, demonstrates that activity of the mutant lac promoter is not affected by LacI or varying levels of IPTG, and our promoter is indeed constitutively active.

Our modified lac promoter, along with downstream EGFP, has been cloned into the submission vector pSB1C3 and submitted to the Registry of Standard Biological Parts as BBa_K311002 (Figure 2). Before submission, DNA sequencing was performed to confirm the sequence of the mutated lac promoter.

Figure 1. Results of Fluorescence assisted cell sorting flow cytometry of E. coli transformants expressing EGFP. The fluorescence is on the horizontal axis while the percentage of events or percentage of cells counted is on the vertical axis. Red, blue and green peaks represent florescence of GFP transformed DH5α pro cells grown at 0 mM, 0.5 mM, and 1 mM Isopropyl IPTG, repectively. The orange and cyan peaks represent the florescence of GFP tranformed JM109 cells grown at 0 mM and 1 mM IPTG. As expected, the peaks for the JM109 transformants had similar peaks because this strain does not express LacI which is the Lac Operon repressor protein that is inactivated by IPTG. Conversely, DH5α pro cells expressed the wild-type LacI. The conformity among the peaks for the wild-type cells suggest that LacI does not bind to the promoter sequence for GFP and it is hence constitutive. Notice the peaks associated with either cell type share similar numbers of events while the peaks from the different cells do not. The difference in events is likely due to the differential GFP expression between the two E. coli strains.

Figure 2. pSB1C3 Plac GFP

Charecterization of Registry Parts submitted by other teams

For fulfilling the judging criteria, apart from our main project we decided on characterizing other parts that already exist in the registry. We have picked up few parts from the registry, described in the table. We are submitting our constructs to the registry; all our constructs are in the biobrick plasmid pSB1C3. We also characterized our promoter (constitutive lac promoter) in the required BioBrick before submitting it to the registry. Our experimental details are described here in detail.

We looked for the parts in the registry that were not previously characterized. We decided to look mainly for regulatory parts, as we wanted to compare strength of different promoters by reporter assays. We finally zeroed down on a family of constitutive promoters that were isolated from a combinatorial library by iGEM Berkeley team in 2006. Further search and sequence alignment revealed that these are mutant tet promoters. In our lab we have few tet promoters which our lab colleagues regularly use for different experiments. We were tempted to compare the tet promoters from this library to our tet promoter. We decided to study one weak and one strong promoter. We picked the plasmid DNA for the part Bba_J23118 and part Bba_J23105 from the 384 well plates, which was supplied to us by the iGEM. We first resuspended the DNA from the plate A, well #20 (J23105) and well #22 (J23118). We transformed the resuspended DNA into TOP 10 cells. Both the plasmids are Ampicillin resistant. We got ~100 transformants the next day. We inoculated single colony from these freshly transformed plates into 50 ml LB media having ampicillin. Next day we isolated both the plasmids to proceed with further cloning. We digested both the plasmid with EcoR I and Pst I enzyme to get the Promoter+RFP part or PoPS generator part. We ligated each promoter+RFP part into the pSB1C3 plasmid backbone (cut by the same restriction enzymes). The transformation of this ligase mix was done in Top10 cells and clones were confirmed by isolating the plasmids from the recombinant cells. These plasmids were subjected to restriction digestion by EcoR I and Pst I enzymes to look for the correct insert size. The biobrick specific primers (VF and VR primers) were used for PCR amplification of the insert, we borrowed the primer sequence from the iGEM registry. We also sequenced few plasmids to make sure that there are no errors in nucleotide sequence and we are using the correct promoter. Our sequencing results for 2 of the clones for both the promoters matched with the sequences given in the registry so we decided to proceed with the promoter activity assays.

We transformed our new plasmids into DH5αPro cells, which constitutively express TetR and LacR (the cell has genome integrated copies of tetR and lacR) and in TOP10 cells (tetR and lacR negative cells). Single colony from each plate was inoculated and culture was allowed to grow overnight in LB medium with chloramphenicol. Next day DH5 alpha pro cells were inoculated in fresh LB medium having varying inducer (aTc) concentration. The inducer concentration was 0, 100 and 200 ng/ml aTc. Top10 cells were also inoculated without addition of inducer.

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In vivo RFP fluorescence was measured using a Becton Dickinson FACS Calibur flow cytometer equipped with a 488 nm argon laser and a 585-610 nm emission filter (FL2) at low flow rate. Fluorescence for all the samples was recorder 6 hours post induction. Flow cytometry allowed us to pick out the population of living cells and determine the amount of RFP that each cell was producing. For each sample, 50,000 events were collected and analyzed using FlowJo software (BD Biosceinces), (figure 1 and 2).

As it is clear from the figures, the inducer concentration has no effect on the fluorescence intensity. For all the inducer concentration the fluorescence remains more or less same for the individual promoters, hence the promoters are constitutive. We calculated the geometric mean for the red fluorescence by FlowJo software, it is interesting to see that J23105 on an average shows a mean fluorescence value of ~21.0 which is half of the mean fluorescence values of J23118 promoter (, ~51.0, Figure.3). The difference in both the promoters was very visible by naked eye. It could be seen on plates and growing liquid cultures, we could not resist clicking a photograph of this clean result. Therefore we conclude that the promoters from the combinatorial library have different characteristics, some of them are weak (Bba_J23105) and some of them are strong (Bba_J23118). It would be worth trying these two promoters in a system, which requires relative expression of certain genes in the same system.

References

Schmidt-Dannert, C., D. Umeno and F. Arnold. "Molecular breeding of carotenoid biosynthetic pathways." Nature biotechnology 18.7 (2000):750-753.