The Coliguard - Differentiation

Introduction

Cell differentiation process can be defined as a “Progressive restriction of the developmental potential and increasing specialization of function that leads to the formation of specialized cells, tissues, and organs”, according to NCBI’s MeSH Database(1). Generally, it is a process by which a cell acquires a new morphological/functional type, capable of performing different and brand new tasks. In most multicellular organism, however, we might find a kind of differentiation that results in a type of cell providing support for another one(2). The differentiation mechanism chosen for our project relies on this perspective, and the first attempt at its implementation as a synthetic biology tool was made by the Paris 2007 iGEM team(3).

The differentiation system adopted for our project was designed focusing the need of creating two subpopulations with distinct characteristics and, mainly, the need of controlling the proportions between both subpopulations. Therefore, we need a differentiation system whose rate could be controlled.

Accordingly to our project objectives, the final results of this differentiation system must be:

• A Worker subpopulation The one responsible for producing the industry compounds of interest, being unable to recognize and destroy contaminants present in the culture medium. In an attempt to maximize productivity, it would be interesting to keep the largest proportion of this subpopulation into the media.

• A Killer subpopulation The one responsible for recognizing and destroying contaminants present in the culture media. This subpopulation also has productivity capacity, yet it could be much lower than the Worker subpopulation since a part of its energetic metabolism must be relocated for maintaining the recognition and destruction systems. To guarantee maximum efficiency of our system, this subpopulation must remain in a basal low proportion, which would be greatly increased in the presence of contaminants. Once they are fully eliminated, our basal proportions must be automatically restored.

The mechanism

In order to design the suggested differentiation system, we will need to modify the expression pattern of several genes, introduce new constructions, and associate two different systems previously proposed by iGEM teams: the Slippage random mechanism by Caltech 2008(4) and the Cre-recombinase’s Paris 2007(3).

The slippage mechanism taking control of basal proportions

Slippage is just one kind of the numerous DNA Polymerase’s mistakes, by which the number of tandem repeats of a determined sequence (AGTC) could be changed during transcriptional process. Our construction will consist of 10 repetitions of AGTC, with an ATG start codon on the upstream position and the Cre-recombinase coding gene on the downstream position, everything under control of a constitutive promoter. In mostly cases, when slippage mistake doesn’t occurs, the (AGTC)10 wouldn’t let the ATG and Cre-recombinase into the same reading frame, hence resulting in no expression of this recombinase. The absence of this expression, found in mostly cases, would result in the Worker cells characteristics, through mechanism that will be explained later. As for the few cases in which the slippage occurs, the number of repetitions could easily turn from 10 to 9. This would let the ATG and Cre-recombinase gene into the same reading frame, thus resulting in Cre-recombinase’s expressions and into Killer cells differentiation.

How Cre-recombinase’s expression would result into Killer cells differentiation?

Cre-recombinase is an enzyme described to produce an unidirectional recombination between lox66 and lox71 recombination sites(5). Therefore, we intend to suit Paris 2007’s construction cassette flanked by both lox sites, in order to insert genes of our interest into it. According to the characteristics of our two subpopulations, the following genes might be necessary to be included into this construction:

• GFP and Resistance Genes: as a reporter, necessary only to assure whether or not the construction is inserted into genomic DNA.

• ftsK: essential gene on maintaining the full cell cycle, whose importance in our project will be later explained.

• finOP: conjugation inhibition system, related with the recognition and destruction cell capacity. Therefore, when we had the absence of Cre-recombinase expression (mostly cases), the resulting cell may has this construction fully operating, hence resulting in a cell capable of maintaining its cell cycle and unable to conjugate (Worker cell’s characteristics). The expression of Cre-recombinase, on the other hand, results in the cleavage of this entire construction, hence resulting in a cell unable to maintain its cell cycle but capable of conjugate (Killer cell’s characteristics).

Why this difference related to conjugation capacity?

Conjugation plays an important role in recognition and destruction systems (see the specific sections for further information). Seeing that, it’s quite important to inhibit conjugation on Worker cells since it must not be able to recognize and eliminate contaminants. The conjugation inhibition will involve a system called finOP. Briefly, this system consists of an antisense RNA (finP) and a small protein (finO)(9). The antisense RNA prevents traJ transcription, which lies immediately upstream of the operon and, in turn, is essential in activating the entire tra operon transcription(10). As for finO, it binds to finP and traJ, thereby allowing the duplex formation(11). Thus, for the correctly function of this system, both finO and finP must be expressed.

Why can’t Killer cells complete their cell cycle?

The inability of completing cell cycle works as a security device, as it wouldn’t let the Killer cells to proliferate and take over the entire culture, thus resulting in loss of productivity. This security system works in two ways: (1) It maintains a low basal proportion of Killer cells, in the absence of contaminants. (2) It guarantees a quickly return to the basal proportion after the elimination of contaminants. As discussed previously, it’s quite important that the proportion of Killer cells could be increased in the presence of contaminants. On the other hand, it’s even more important that this proportion could quickly be restored to the basal levels since the contaminants were eliminated. The inability of completing cell cycles makes the Killer cells proportion directly and almost exclusively dependent to the specific stimulus trigged by the presence of contaminants. Thus, as soon as this stimulus ceases, the proportion of Killer cells would be drastically reduced since they are unable to reproduce and proliferate

How the basal proportion will change in the presence of contaminants?

The answer is AI2 (auto-inducer 2). AI2 is a member of signaling molecules used in bacteria quorum sensing, produced by both gram-positive and gram-negative bacteria(6, 7 and 8). We thought that, since we could silence the AI2 coding gene on our lineage, any detectable presence of this molecule would be due to the presence of contaminants, thereby working as a trigger signal. Thus, we decided to place the Cre-recombinase coding gene under the control of a promoter with sensibility to AI2. We will use AI2 self promoter for that purpose, since there is a negative feedback control related to AI2 production. In short, when contaminants are present they probably will produce AI2, which will trigger Cre-recombinase expression. This expression, as previously discussed, is responsible for Killer cells differentiation.

Strategy

As we previously explained, the model that we´ve developed to the differentiation subpart of our project involves the modification and coupling of two strategies previously used by the teams of Paris 2007 and Caltech 2008. The Paris 2007 team used the FX58 E. coli strain, which has the construction Lox71-FSTK-Lox66 inserted in its genome. The FSTK gene is essential to cell division; its disruption stops cell division. The Lox71 and Lox66 sites are cleaved by a CRE recombinase, excising them and all the DNA sequences located between them. In Paris model, the Cre recombinase gene is under the control of dapA promoter. (for more information about Paris 2007 project, access the link: http://parts.mit.edu/igem07/index.php/Paris) The Caltech 2008 team inserted the repetitive sequence AGCT10 between the start codon of the GFP gene and the rest of its ORF. In this case, the ORF is not in frame with its start codon, so the GFP is not corrected translated. However, DNA polymerase may slip when it replicates repetitions of small nucleotide polymers. So, during the replication of this construction, in some cases the AGCT10 is replaced by AGCT9. In this case, the GFP ORF is in frame with its start codon, and the protein is corrected translated. In our model, we aimed to insert two constructions flanking the FSTK gene; a) an upstream construction: LOX71-GFP; and b) a downstream construction: FinO-FinP-Lox66. The presence of finO and FinP genes impede the cell to conjugate. Moreover, we aimed to insert the repetition AGCT10 between the Cre recombinase start codon and the rest of its ORF; when Cre recombinase ORF is in frame with the start codon, it will be translated and act on the Lox based construction, excising the FSTK, FinO and FinP genes; so, the cell will lose its division skills, however it will be able to conjugate.

Constructions

The constructions we´ve developed to this part of the project are described below.

1) BBa_K284043: AGTC repetition and CRE recombinase under constitutive promoter After the amplification of CRE recombinase by PCR, we cloned it into the PSB1A3 vector, according to the standard assembly strategy, resulting in the biobrick BBa_K284031. The following steps were also based on the standard assembly strategy, gathering the biobricks BBa_R0040 (pTet promoter), BBa_284040 (AGCT repetition), BBa_284031 (Cre recombinase without the start codon) and BBa_B0015 (double terminator), resulting in BBa_K284043.

2) BBa_K284090: Downstream construction flanking FSTK gene This construction was developed to be inserted in the downstream region of the FSTK gene. Lox 71 is a cleavage site for CRE recombinase. This biobrick was assembled according to the standard protocol, gathering the previously existing biobricks BBa_I718017 (Lox 71), BBa_R0040 (pTet promoter), RBS (BBa_B0030), BBa_E0040 (green fluorescent protein ORF) and BBa_B0015 (double terminator).

3) Upstream construction flanking the FSTK gene

This construction is a composite part formed by the biobricks BBa_K284060, BBa_K284071 and BBa_I718017.

3.1) BBa_K284060: FinP under constitutive promoter

After the amplification of FinP (from E. coli F plasmid) by PCR, we cloned it into the PSB1A3 vector, according to the standard assembly strategy, resulting in the biobrick BBa_K284033. The following steps were also based on the standard assembly strategy, gathering the biobricks BBa_R0040 (pTet promoter), BBa_B0030 (RBS), BBa_284033 (FinP form F plasmid) and BBa_B0015 (double terminator), resulting in BBa_K284060.

3.2) BBa_K284071: FinO under constitutive promoter After the amplification of FinO (from E. coli R100 plasmid) by PCR, we cloned it into the PSB1A3 vector, according to the standard assembly strategy, resulting in the biobrick BBa_K284032. The following steps were also based on the standard assembly strategy, gathering the biobricks BBa_R0040 (pTet promoter), BBa_B0030 (RBS), BBa_284032 (FinO form R100 plasmid) and BBa_B0015 (double terminator), resulting in BBa_K284071.

3.3) BBa_K284080: Upstream construction flanking FSTK gene This construction was developed to be inserted in the upstream region of the FSTK gene. Lox 66 is a cleavage site for CRE recombinase. This biobrick was assembled according to the standard protocol, gathering the biobrick BBa_K284060 (FinP under constitutive promoter), BBa_K284071 (FinO under constitutive promoter) and BBa_I718016 (Lox 66).

REFERENCES

1. http://www.ncbi.nlm.nih.gov/sites/entrez?db=mesh

2. Weismann, A. The Germ Plasm. 1892

3. http://parts.mit.edu/igem07/index.php/Paris

4. https://2008.igem.org/Team:Caltech

5. Zhang, Z. and Lutz, B. Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. Nucleic Acid Research.

6. Cao, J.G. & Meighen, E.A. J Biol Chem 264, 21670-21676 (1989)

7. Miller, S.T. et al. Mol Cell 15, 677-687 (2004)

8. Miller, M. B.; Bassler, B. L. Annu. Rev. Microbiol. 2001, 55, 165-199

9. Gubbins, M.J et al. J. Biol Chem 278, pp. 27663–27671

10. van Biesen, T. et al. Mol Microbiology 10, 35-43

11. Koraimann, G. et al. Mol. Microbiology 21, 811-821