Team:Waterloo/Project

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Project Aim and Rationale

There are many useful bioproducts that can be efficiently expressed in vivo but are either cost-prohibitive or impossible to synthesiize and purify in vitro. To address this need for a means of manufacturing such bioproducts, a great deal of work had been done to study [http://openwetware.org/wiki/Minicells minicells], genome-free sacks of cytoplasm that bud off from certain mutant bacterial strains, and from which plasmid-driven expression has previously shown to be possible. Minicells showed substantial promise for possible therapeutic applications since they could generate useful products without replicating themselves. However, for in situ delivery of their products, they must be filtered from the live bacteria from which they were released.

Transient plasmid-driven expression in minicells has been shown to last for several hours, and though this "lifetime" can be improved by use of a nutrient bath, it is limited by cytosolic resources. Once these resources are exhausted, the minicell becomes inactive and degrades.

Our project improves on the two issues described above. A bacterial cell already containing a plasmid encoding bioproduct synthesis genes, will self-destruct by degrading its own genome and transiently produce the bioproduct until cell resources have been exhausted. Having the original bacteria serve as the factory means that not only is there no live parental budding strain to purify from the culture, but also more cell resources are available to express larger quantities of bioproduct.

Genome degradation is achieved using the combined activity of a restriction endonuclease to fragment the genome and an exonuclease to hasten degradation. The gene for the protein of interest will be located on a plasmid lacking recognition sites for the endonuclease, allowing it to remain intact after genomic degradation. The plasmid genes will be expressed using the remaining cell resources until they expire. The primary application of this design would be an in situ compound production and delivery system for agricultural, industrial or therapeutic use.

Project Design

Production and Regulation of Nuclease Genes

Endo- and exonucleases will be cloned between the lambda pR promoter and the Plac promoter. The lambda promoter will drive the production of the forward transcript for translation of the genes (which are toxic), while pLac will drive production of antisense transcripts to maximize repression of the two nucleases during uninduced conditions. Cultures will initially be grown on media containing IPTG. Expression of the unstable cI repressor (C0051) will be driven by another instance of a lacI repressable promoter. Since nuclease production is usually repressed, endogenous lacI production should be sufficient. Any antisense transcripts from the leaky Plac will not be able to compete with the strong lambda promoter during expression of the nucleases.

Construction Strategy

Biobrick Part Number Legend

[TT] BBa_B0015
[Pr] BBa_R0051
[Plac] BBa_R0011
[CI] BBa_C0051
[T7gene6] BBa_K093001
[PmeI] BBa_K093002

Steps in Assembly

T7 gene 6 exonuclease will be amplified using PCR with primer-adapters for the BioBrick Prefix and Suffix.
Pseudomonas mendocina PmeI endonuclease will be synthesized with its ORF flanked by BioBrick Prefix and Suffix.

A nuclease construct will be constructed using crossover PCR of the nuclease gene.

A convergent promoter system designed to flank the nuclease genes are denoted below by Construct 1 and Construct 2. Construct 2 will be added downstream of the nuclease constructs such that no sense transcript and protein will be made at this stage. Construct 1 will be added upstream in the next round of assembly. This will ensure repression of the system in the presence of CI repressor and IPTG.

Construct 1
Construct 2

The addition of Construct 2 is a non toxic intermediate step.

The addition of Construct 1 ensures for proper repression in the presence of IPTG and the CI repressor.

The already assembled CI repressor under the control of Plac must be cotransformed and plated on IPTG

The final independent construct:

These tests will determine:
- The length of time needed for the destruction of the genome by nuclease expression
- The optimal integration site based on the ideal length of time for nuclease gene production.
- Where the REN sites are on the genome.
- The rate of degradation by the exonuclease.

Note:

Ligation scars are not shown.
Ribosome binding sites are present for open reading frames (T7gene6, PmeI, CI) upstream in the forward orientation.
[forward arrow] denotes forward orientation
[reverse arrow] denotes reverse position.

Implementation

Preliminary Assay of Genomic Degradation

Chassis will be transformed with the preliminary construct.
Uninduced cell growth will be assayed to determine if the repression is strong enough to allow for the uninhibited growth of uninduced cells.
Cells will be induced for different periods of time. The induction will be terminated by washing to eliminate IPTG. Assay for sterility of culture growth test with positive and negative controls (uninduced transformants and no inoculation, respectively). Genome destruction and plasmid safeness will be checked by genomic and plasmid preps of the induced culture. [(looking for smear and nice band?)]

Integration of Nuclease Genes

The genome degradation construct will be integrated into the genome via a proposed standard integration vector.
The vector will contain a BioBrick cloning site and unique double digest sites flanking the BioBrick cloning site and suitable elements for selection (Sac B and antibiotic resistance). The unique double digest sites will allow the user to customize the regions of homology for a double crossover homologous recombination.

The integration vector will be a vector with the oriR6k origin of replication that only replicates in E. coli lambda pir lysogen. For integration, the plasmid will be transformed into a strain of E. coli that is not a lambda pir lysogen by selecting for recombination using the antibiotic respective to the vector. The vector will be a suicide vector and therefore in any non lambda pir strain, a single recombination will be selected. The cells will then be removed from selective pressure to allow for the next recombination to occur. The double recombinants will be selected using sucrose, since the vector will also contain a sacB gene, which confers sucrose sensitivity. The putative double recombinants must then be screened for the respective antibiotic sensitivity.

Safe Target Plasmid

In order to ensure expression of a desired gene post degradation of the genome, the plasmid must be free of recognition sites for the restriction endonuclease PmeI and therefore be unable to be influenced by the exonuclease. Red fluorescent protein is used as a reporter gene.

The reporter will be put under the control of the LexA repressible promoter. The genome degradation method produces single stranded DNA due to the presence of the 5’-3’ [(check gene 6 exo activity direction to confirm)] exonuclease. When there is single stranded DNA in the cell, RecA is upregulated as part of the SOS response, and RecA cleaves lexA. Therefore when the genome degradation is induced, the expression of the gene of interest will be subsequently induced. For this reason, the final construct must be integrated into a RecA + strain (TG1 is Rec+ and lacIq, and will work completely since lacI is required for the induction of the genome degradation operon).

The plasmid safeness will be assayed when exposed to the nucleases. From that, reporter production can be evaluated, and the induction time relative to the time of degradation can be optimized.

Preliminary Testing of Promoter Reporter Constructs

A preliminary experiment was conducted to test the expression of the pRecA promoter with LexA binding sites (K093000), the constitutive promoter pconst (J23118) and PocI, the lambda Pr promoter containing the operator for lambda cI repressor (K093010). Visual observations of liquid cultures show that expression of RFP under the control of the PocI promoter in absence of the repressor is high and similar to expression under control of the constitutive promoter. These cultures were all extremely pink, and also form pink colonies on solid media. The uninduced pRecA culture shows very low levels of expression and light pink colonies.

Cultures were inoculated in triplicate. Saturated overnight cultures were inoculated from a single colony. A second set of cultures were inoculated and grown to an OD600 of approximately 0.8. For pRecA, 3 conditions were set and conducted at both cell concentrations; uninduced culture, culture exposed to 125V of UV culture exposed to 250V of UV. To provide consistency for these exposures, sample was taken from the same culture at each level of exposure.

Readings were taken using a microplate reader, each sample plated in triplicate. The excitation peak and emission peaks of RFP (E1010) at 584 nm and 607 nm respectively were used for the readings. Saturated culture and culture at an OD600 of approximately 0.8 with no RFP expression were used as negative controls.

These preliminary tests resulted in very high background readings and results that were not statistically significant as a result of this signal to noise ratio (data not shown). Repetition and protocol modification is required to continue these tests. UV seemed to have little to no effect on the induction of the pRecA promoter. Other crosslinking agents will be tested, including mitomycin C, in addtion to testing other levels of UV exposure. PocI will also be tested in the presence of the cI repressor.

Future Directions and Applications

A post induction of genome degradation freeze drying system can be utilized to enable storage and transport for subsequent thawing and induction of the desired product.
Quorum sensing control T7PoI can be expressed. The nuclease operon will be put under T7 control instead of TetR

References

O'Connor,C.D., & Timmins, K.N. (1987). Highly repressible expression system for cloning genes that specify potentialy toxic proteins. Journal of Bacteriology. 169, 4457-4462.

GUZMAN, L, BELIN, D, CARSON, M. J., & BECKWITH, J (1995). Tight Regulation, Modulation, and High-Level Expression by J. of Bacteriology, 177, 4121-4130.

Macdiarmid, et al. Cancer Cell 11, 431-445, May 2007

@@ Line 1: / Line 1: @@
 <!--- The Mission, Experiments --->
-{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
+{| style="color:#1b2c8a;background-color:#FBCC30;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
 !align="center"|[[Team:Waterloo|Home]]
 !align="center"|[[Team:Waterloo/Team|The Team]]
@@ Line 11: / Line 11: @@
 |}
-== '''Project Aim''' ==
+<br>
-Our system is designed as a genome-free, cell-based expression system capable of producing a desired protein or activating a pathway in response to environmental signals. Genome degredation is achieved using the combined activity of a restriction endonuclease to fragment the genome and an exonuclease to hasten degredation. The gene for the protein of interest will be located in a plasmid lacking recognition sites for the endonuclease, allowing it to remain intact after genomic degredation. The plasmid genes will be expressed using the remaining cell resources until they expire. The primary application of this design would be an in situ compound production and delivery system for agricultural, industrial or therapeutic use.
+== '''Project Aim and Rationale''' ==
-[[Image:2008Design.png]]
+There are many useful bioproducts that can be efficiently expressed <i>in vivo</i> but are either cost-prohibitive or impossible to synthesiize and purify <i>in vitro</i>. To address this need for a means of manufacturing such bioproducts, a great deal of work had been done to study [http://openwetware.org/wiki/Minicells minicells], genome-free sacks of cytoplasm that bud off from certain mutant bacterial strains, and from which plasmid-driven expression has previously shown to be possible. Minicells showed substantial promise for possible therapeutic applications since they could generate useful products without replicating themselves. However, for <i>in situ</i> delivery of their products, they must be filtered from the live bacteria from which they were released.
-== Design Details ==
+Transient plasmid-driven expression in minicells has been shown to last for several hours, and though this "lifetime" can be improved by use of a nutrient bath, it is limited by cytosolic resources. Once these resources are exhausted, the minicell becomes inactive and degrades.
+Our project improves on the two issues described above. A bacterial cell already containing a plasmid encoding bioproduct synthesis genes, will self-destruct by degrading its own genome and transiently produce the bioproduct until cell resources have been exhausted. Having the original bacteria serve as the factory means that not only is there no live parental budding strain to purify from the culture, but also more cell resources are available to express larger quantities of bioproduct.
+Genome degradation is achieved using the combined activity of a restriction endonuclease to fragment the genome and an exonuclease to hasten degradation. The gene for the protein of interest will be located on a plasmid lacking recognition sites for the endonuclease, allowing it to remain intact after genomic degradation. The plasmid genes will be expressed using the remaining cell resources until they expire. The primary application of this design would be an in situ compound production and delivery system for agricultural, industrial or therapeutic use.
+== Project Design ==
+[[Image:2008Design.png]]
 === Production and Regulation of Nuclease Genes ===
-*T7 gene 6 exonuclease and Psuedomonas mendocina PmeI endonuclease will be amplified using PCR with primer-adapters for the biobrick prefix & suffix.
+*Endo- and exonucleases will be cloned between the lambda pR promoter and the Plac promoter. The lambda promoter will drive the production of the forward transcript for translation of the genes (which are toxic), while pLac will drive production of antisense transcripts to maximize repression of the two nucleases during uninduced conditions. Cultures will initially be grown on media containing IPTG. Expression of the unstable cI repressor (C0051) will be driven by another instance of a lacI repressable promoter. Since nuclease production is usually repressed, endogenous lacI production should be sufficient. Any antisense transcripts from the leaky Plac will not be able to compete with the strong lambda promoter during expression of the nucleases.
-*REN and exonuclease will be cloned between the lambda pR promoter and the Plac promoter. The lambda promoter will drive the production of the forward transcript for translation of the genes (which are toxic), while pLac will drive production of antisense transcripts to maximize repression of the two nucleases during growth. Cultures will initially be grown on minimal media with lactose as the sole carbon source. The unstable cI repressor (BBa_C0051) will be in our construct driven by another instance of a lacI repressable promoter. Since nuclease production is usually repressed, endogenous lacI production should be sufficient. Any antisense transcripts from the leaky Plac will not be able to compete with the strong lambda promoter during expression of the nucleases.
 ==Construction Strategy==
 ===Biobrick Part Number Legend===
-[ !!!!Add descriptions of these parts as well as anything that is missing, or link to parts page]
 *[TT] BBa_B0015
 *[Pr] BBa_R0051
 *[Plac] BBa_R0011
 *[CI] BBa_C0051
-*[T7gene6] ____
+*[T7gene6] BBa_K093001
-*[T7gene5] _____
+*[PmeI] BBa_K093002
-*[PmeI] _____
 ===Steps in Assembly===
+*T7 gene 6 exonuclease will be amplified using PCR with primer-adapters for the BioBrick Prefix and Suffix.
+*''Pseudomonas mendocina'' PmeI endonuclease will be synthesized with its ORF flanked by BioBrick Prefix and Suffix.
-*A <b>nuclease construct</b> will be constructed from standard assembly of each nuclease gene.
+*A <b>nuclease construct</b> will be constructed using crossover PCR of the nuclease gene.
-[[Image:1Sequence_(1).jpg]]
+[[Image:Sequence_(1).jpg]]
 *A <b> convergent promoter system</b> designed to flank the nuclease genes are denoted below by Construct 1 and Construct 2. Construct 2 will be added downstream of the nuclease constructs such that no sense transcript and protein will be made at this stage. Construct 1 will be added upstream in the next round of assembly. This will ensure repression of the system in the presence of CI repressor and IPTG.
-[[Image:1Sequence_(2).jpg]] Construct 1<br>
+[[Image:Sequence_(2).jpg]] Construct 1<br>
-[[Image:1Sequence_(3).jpg]] Construct 2
+[[Image:Sequence_(3).jpg]] Construct 2
 *The <b>addition of Construct 2</b> is a non toxic intermediate step.
@@ Line 69: / Line 76: @@
 Note:
 *Ligation scars are not shown.
-*Ribosome binding sites are present for open reading frames (T7gene6, T7gene5, PmeI, CI) upstream in the forward orientation.
+*Ribosome binding sites are present for open reading frames (T7gene6, PmeI, CI) upstream in the forward orientation.
 *[forward arrow] denotes forward orientation
 *[reverse arrow] denotes reverse position.
 ==Implementation==
-===Preliminary Assay of Genomic Degredation===
+===Preliminary Assay of Genomic Degradation===
 *Chassis will be transformed with the preliminary construct.
 *Uninduced cell growth will be assayed to determine if the repression is strong enough to allow for the uninhibited growth of uninduced cells.
@@ Line 81: / Line 88: @@
 ===Integration of Nuclease Genes===
 *The genome degradation construct will be integrated into the genome via a proposed standard integration vector.
-*The vector will contain a biobrick cloning site and unique double digest sites flanking the biobrick cloning site and suitable elements for selection (Sac B and antibiotic resistance). The unique double digest sites will allow the user to customize the regions of homology for a double crossover homologous recombination.
+*The vector will contain a BioBrick cloning site and unique double digest sites flanking the BioBrick cloning site and suitable elements for selection (Sac B and antibiotic resistance). The unique double digest sites will allow the user to customize the regions of homology for a double crossover homologous recombination.
-[DIAGRAM]
+*The integration vector will be a vector with the oriR6k origin of replication that only replicates in ''E. coli'' lambda pir lysogen. For integration, the plasmid will be transformed into a strain of ''E. coli'' that is not a lambda pir lysogen by selecting for recombination using the antibiotic respective to the vector. The vector will be a suicide vector and therefore in any non lambda pir strain, a single recombination will be selected. The cells will then be removed from selective pressure to allow for the next recombination to occur. The double recombinants will be selected using sucrose, since the vector will also contain a ''sacB'' gene, which confers sucrose sensitivity. The putative double recombinants must then be screened for the respective antibiotic sensitivity.
-*The integration vector will be a vector with the oriR6k origin of replication that only replicates in E. coli [lambda] pir lysogen. For integration, the plasmid will be transformed into a strain of E. coli that is not a [lambda] pir lysogen by selecting for recombination using the antibiotic respective to the vector. The vector will be a suicide vector and therefore in any non [lambda] pir strain, a single recombination will be selected. The cells will then be removed from selective pressure to allow for the next recombination to occur. The double recombinants will be selected using sucrose, since the vector will also contain a <i>sacB</i> gene, which confers sucrose sensitivity. The putative double recombinants must then be screened for the respective antibiotic sensitivity.
 ===Safe Target Plasmid===
@@ Line 95: / Line 100: @@
 *The plasmid safeness will be assayed when exposed to the nucleases. From that, reporter production can be evaluated, and the induction time relative to the time of degradation can be optimized.
-==Future Directions and Applications==
+==Preliminary Testing of Promoter Reporter Constructs==
-*A post induction of genome degredation freeze drying system can be utilized to enable storage and transport for subsequent thawing and induction of the desired product.
-*Quorum sensing control T7PoI can be expressed. The nuclease operon will be put unter T7 control instead of TetR
+A preliminary experiment was conducted to test the expression of the pRecA promoter with LexA binding sites (K093000), the constitutive promoter pconst (J23118) and PocI, the lambda Pr promoter containing the operator for lambda cI repressor (K093010). Visual observations of liquid cultures show that expression of RFP under the control of the PocI promoter in absence of the repressor is high and similar to expression under control of the constitutive promoter. These cultures were all extremely pink, and also form pink colonies on solid media. The uninduced pRecA culture shows very low levels of expression and light pink colonies.
+Cultures were inoculated in triplicate. Saturated overnight cultures were inoculated from a single colony. A second set of cultures were inoculated and grown to an OD600 of approximately 0.8. For pRecA, 3 conditions were set and conducted at both cell concentrations; uninduced culture, culture exposed to 125V of UV culture exposed to 250V of UV. To provide consistency for these exposures, sample was taken from the same culture at each level of exposure.
+Readings were taken using a microplate reader, each sample plated in triplicate. The excitation peak and emission peaks of RFP (E1010) at 584 nm and 607 nm respectively were used for the readings. Saturated culture and culture at an OD600 of approximately 0.8 with no RFP expression were used as negative controls.
+These preliminary tests resulted in very high background readings and results that were not statistically significant as a result of this signal to noise ratio (data not shown). Repetition and protocol modification is required to continue these tests. UV seemed to have little to no effect on the induction of the pRecA promoter. Other crosslinking agents will be tested, including mitomycin C, in addtion to testing other levels of UV exposure. PocI will also be tested in the presence of the cI repressor.
+==Future Directions and Applications==
+*A post induction of genome degradation freeze drying system can be utilized to enable storage and transport for subsequent thawing and induction of the desired product.
+*Quorum sensing control T7PoI can be expressed. The nuclease operon will be put under T7 control instead of TetR
-Should we still keep all of this (see below??)
-(???)
-*Already have RFP w/ RBS and TT - should be able to just stick pLac (e .g.) in front of this for preliminary verification. GFP may be a better option for later testing of expression post-kill due to its faster turnover giving better temporal resolution.
-*If we did this, we could also use this (in a lacIq background, maybe?) as a preliminary form of control, WT (or lacIq) cell + reporter plasmid (pLac+rfp)
-**Test reporter by IPTG induction
-**WT (or lacIq) cell + reporter plasmid with nuclease operon (e. g. under TetR control- have some semi-constructed stuff for this, tight enough control for growth?)
-**Test for reporter expression upon expression of nuclease (aTe derepression of pTet-controlled nuclease operon)
-*Later, could have quorum sensing control T7PoI expression (q.s. + T7 stuff may already exist together in the registry?), and put the nuclease operon under T7 control (instead of TetR)
-== Tentative Stages of Construction ==
-'''Stage 1:'''
-*Obtain gene 6 from T7 or gene D15 from T5 bacteriophage and a suitable REN gene from Psuedomonas mendocina (PmeI) or Psuedomonas alcaligenes (PacI) via PCR with primer-adapters for the biobrick prefix & suffix. T7 may be easier to obtain, and we have been advised that unless we can think of a really good reason to use T5 it isn't worth the trouble of getting T5 over T7.
-*To start on the genome degradation side, we can clone the REN (considering PmeI, PacI)and the exonuclease (T5 D15, or T7 gene6)between the lambda pR promoter driving the production of the proper transcript for translation of the genes (which are toxic), and on the other side,pLac (or another lacI repressable promoter) driving production of antisense transcripts to maximize repression of the two nucleases during growth on minimal media with lactose as the sole carbon source. The the unstable cI repressor (BBa_C0051) will be in our construct driven by another instance of a lacI repressable promoter. It should be sufficient to rely on endogenous lacI considering we want induction of pLac at most times, and during production of the nucleases, to destroy the genome, a few antisense transcripts leaking from pLac should not be a problem with the strong lambda promoter making sense transcripts at such a high  rate relative to the repressed pLac.
-*Transform chassis with the preliminary construct.
-*Assay uninduced cell growth. Is our repression strong enough to allow uninduced cells to grow uninhibited?
-*We induce for different periods of time (turn off by transferring from lactose to glucose, or glucose to arabinose, depending on system) and assay for genome destruction by a growth test with positive and negative controls (uninduced transformants and no inoculation, respectively); we also take an aliquot, purify the DNA, and run the DNA on a gel alongside the uninduced cells and chassis carrying untransformed plasmid.
-**Aside: The tests used to prove sterility by the minicell people for their minicell preparation were plating on growth agar plates, incubating at 37 overnight, and checking for absence of colonies; also inoculating thioglycolate broth with the preparations and incubating for 14 days to demonstrate the absence of any slow-growing organisms (Macdiarmid, et al. 2007)
-*Then we have data on how long the nucleases will need to be expressed to destroy the genome. We give the modelling team some work. We get them to help find out exactly where is the optimal spot to integrate our degradation operon into the E. coli genome, based on how long the nuclease genes need to be on, where the REN sites are on the genome, and the rate of degradation by the T5 exonuclease (that data we have).
-*We can then subject our safe plasmid with the reporter to the two nucleases in vitro and run on a gel. We could buy the nucleases if we have the budget or maybe we could purify some from our cells that make them, but that might be difficult
-'''Stage 1b (in parallel with stage 1):'''
-*Clone reporter in to plasmid of choice.
-*Make sure plasmid has no REN sites for REN in our degradation operon
-*Assay plasmid safeness when exposed to nucleases (exonuclease and REN)
-'''Stage 2:'''
-*Construct integrating plasmid with regions of homology, which we had picked based on degradation rates and suitable elements for selection (SacB and antibiotic resistance). Or obtain commercial integrating plasmid to get our stuff in close enough to it's optimal spot.
-*Select for cells with integrated genes by plating on media with sucrose and antibiotic
-*Assay the viability of uninduced cells again
-*Assay genome degradation in much the same way as before, with appropriate controls.
-'''Stage 2b:'''
-*Put reporter under control of LexA repressible promoter or other inducible/repressible promoter system
-'''Stage 3:'''
-*Transform genome degrading chassis (from stage 2)
-*Induce genome degradation
-*Genome degradation times should be elucidated somewhat, and we should be somewhat able to tell whether or not the reporter is being produced too early (LexA repression). Since both cI and LacI are being used in the degradation regulation, something that will autoregulate would be preferable. There are other options, such as pBAD from that L-ara operon, induced indirectly by arabinose, and not much else.
-*The ultimate would be to induce genome degradation, freeze dry, thaw, then induce the plasmid and have the reporter be made (because any practical application of this is going to require a decent shelf life). If the cells were not viable when they left the "manufacturing" facility, it would be preferable to having to induce genome killing in the field.