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Team:WHU-China/Project
From 2012.igem.org
Project Description
The above figure is the overview of our design
The utmost purpose of our project is to emancipate people from obesity. It can be achieved by genetically modifying a resident intestinal microbe, such as E.coli, to create a novel beneficial bacterial which is competent to eliminate the excessive absorption of calorie.
Fatty acids are our primary targets. To prevent the over in-take and accumulation of fatty acids, we will try to engineer microbes that can metabolize the excessive fatty acids in diet efficiently and effectively before they are absorbed by the host. To achieve this, we will overexpress the enzymes responsible for fatty acid degradation under the control of a natural sensor for concentration of fatty acids-- FadR, a repressor for the genes involved in fatty acid degradation. A constitutive promoter will be fused to the original binding site of FadR. If such a promoter is placed upstream of the target genes, they can only express when the FadR senses the high concentration of fatty acids and slides off the site on DNA. The gene expression will solely respond to concentration of fatty acids.
Also, since the glucose can be transformed into fatty acids in our body, we will try to transform the glucose into polymers, such as cellulose, which cannot further be degraded and absorbed by the host. Instead of contributing to the formation of fatty acids, the glucose is turned into a healthier substance. The polymers made from the glucose may facilitate the growth of other intestinal microbes which have been proved to be beneficial for maintaining a normal weight. Enzymes responsible for cellulose synthesis are accessible from other species of bacteria and can be implanted into E.coli. To sense the glucose concentration and respond exclusively to it, we choose CRP as a regulator. Specifically, we will change this activator into a repressor by overlapping its binding site downstream the constitutive promoter instead of in front of it. Then the relative gene can only be activated when the CRP cannot bind the site on DNA at high glucose concentration. This synthetic promoter may have broad applications. For example, it can be used in gene therapy for diabetes.
Another problem we will try to tackle is the survival and the colonization of the bacteria in intestine. It is commonly accepted that a species introduced to a new enviornment gets the chance of surviving and even being dominant if it can utilize an energy resource that cannot be used by any other species. According to the theory, the unmatchable ability of the E.coli to utilize the fatty acids as its carbon sources can already partially fulfill the goal. The adhesion to intestinal cells is another factor for its survival other than the energy requirement. We will try to increase the adhesion ability of the E.coli by enhancing its production of c-di-GMP, a second massager which has been reported to increase adhesion of bacteria.
Biological safety should be paid equal attention in the progress of designing novel probiotic. To prevent the uncontrolled reproduction of the modified E.coli in the intestine, we have designed a death system to wipe out the GMOs at will. It is designed to exploit the natural sensor for the signal molecule xylose to regulate its target, the endonuclease responsible for killing the cell quietly without setting off any immunological reaction. Also, Horizontal gene transfer will also trigger the death of the recipient.
To sum up, we not only propose a novel and interesting way to tackle the obese problems but also create the biological sensors for fatty acids and glucose which have broad applications in detection and therapy of related diseases.
Obesity: a serve global problem
Obesity refers to a health condition that body fat is accumulated to some extent. According to WHO, body mass index (BMI) is an index of weight-for-height that is commonly used to classify obesity in adults. It is a risk factor for various diseases, such as cardiovascular diseases (mainly heart disease and stroke), type 2 diabetes, musculoskeletal disorders (especially osteoarthritis), some cancers (endometrial, breast, and colon).
As it is shown in figure 1 and 2, a large amount of people from all over the world are overweight in both developed countries and developing contries and it is and will become more and more serve.
Figure 1(from reference [4]): Past and projected prevalence of overweight (BMI ≥25 kg/m2)
Figure 2: Prevalence of obesity in different countries.
(Picture from The Wellington Grey blog)
The Cause of Obesity
Obesity is most commonly caused by a combination of excessive food energy intake, lack of physical activity, and genetic susceptibility, although a few cases are caused primarily by genes, endocrine disorders, medications or psychiatric illness.
However, the problem of obesity emerged globally only several decades ago. Since the change of genome of a species requires a long time, the outbreak of obesity is unlikely to be caused by changes in human genome. For most individuals, controlling food intake and doing physical activity in a proper way are effective strategies to lose weight. But for some people whose health condition or current life pace keeps them away from systemic and regular exercise and dieting, modulate the composition of microorganisms in intestine might act as an alternative.
Reports by Gordon have shown that, apart from human genome, the collective genome of microorganisms (microbiome) in human intestine is associated with our obesity [1]. Furthermore, microbiome is able to be changed through control of food intake [1].
Two groups of beneficial bacteria are dominant in the human gut, the Bacteroidetes and the Firmicutes. The relative proportion of Firmicutes is increased in obese people by comparison with lean people [2].
Figure 3: How excess of energy contributes to obesity
Pertinent study by Gordon attested their initial hypothesis that changes
in microbial component have a causal relationship with obesity, thus might
have potential therapeutic implications [2] [3]. Colonization of germ-free
mice with an ‘obese microbiota’ results in a significantly greater increase
in total body fat than colonization with a ‘lean microbiota’ [3].
Figure from reference [3]
Present strategies to lose weight
Dieting, excercise, Drugs and surgery and major ways to lose weight. However, they all have many drawbacks. Dieting may cause nutritional imbalance and can be a heavy mental burden since the person may not be able to enjoy the food he want. Excercise requires regular time and is ineffective in many cases. Drugs and surgery may have many side effects and are many times costly.
Our idea
Previous situations and insights construct our theoretical fundament.
We try to utilize synthetic biology to provide a cheap, convient, effective
and safe approach for treating obesity. Instead of passive alternation
of microbiota, we are trying to construct an engineered E.coli----- E.coslim
to positively change microbiota in intestine. As Figure 3 shown, we place
E.coslim in the role of sensing and consuming excessive energy, thus leads
to the double effects: lowering the proportion of Firmicutes and increasing
that of Bacteroidetes, and decreasing the energy available in one’s intestine.
To achieve these two goals, we designed four devices, fatty acids consumption, cellulose synthesis, colonization and death device of E.coslim.
References
[1] Ruth E. Ley1, Peter J. Turnbaugh1, Samuel Klein1 & Jeffrey I. Gordon1 Microbial ecology: Human gut microbes associated with obesity. Nature 444, 1022-1023 (21 December 2006)
[2] Peter J. Turnbaugh1, Ruth E. Ley1, Michael A. Mahowald1, Vincent Magrini2, Elaine R. Mardis1,2 & Jeffrey I. Gordon1 An obesity-associated gut microbiome with increased capacity for energy harvest. Vol 444|21/28 December 2006| doi: 10.1038/nature05414
[3] Ley RE. Obesity and the human microbiome. Curr Opin Gastroenterol. 2010 Jan; 26(1):5-11.
[4] Y Claire Wang et.al. Health and economic burden of the projected obesity trends in the USA and the UK. Lancet. 2011
Introduction
Fatty acids and sugar should be the primary targets for genetically engineered probiotics that can help people lose weight. For probiotics to degrade fatty acids and to convert glucose into cellulose, they must be able to sense and be regulated by exsitence of those substrates. Otherwise, the system may not only be not efficient but also may cause serious problem. However, there is no promoter exist in nature that can solely regulated by glucose or fatty acids. Therefore, to achieve our goals, we designed an indirect pathway and a direct synthetic promoter to sense and be regulated by glucose concentration. Also another synthetic promoter was designed to sense and be regulated by fatty acids.
Indirect Pathway Design
In a cell, the total amount of ATP, ADP and AMP molecules remains constant. Low glucose concentration results in high activity of adenylate cyclase converting ATP into cAMP, who binds and converts cAMP receptor protein (abbreviated as CRP) to DNA-binding configuration. Conversely, when glucose concentration gets high, more ATP and less cAMP will be produced, resulting in low DNA-binding activity of CRP.
We embed gene cI of lambda phage(BBa_P0451) downstream promoter PcstA (BBa_K118011) activated by the binding of CRP, and genes of red fluorescence protein(RFP, BBa_I13507) respectively downstream the promoter BBa_R0051 repressed by protein cI. In this way we construct an indirect regulation pathway with sensus glucose, transcription activator CRP and transcription repressor cI. If the device works as design, output of RFP will be increased following the elevation of glucose concentration, and vice versa.
Method
Construction of plasmid for indirect regulation pathway
In this experiment, RFP reported the function of the indirect regulation pathway.
K861173: BBa_I13507, an mRFP generator with RBS and terminator, was embedded after CRP activated promoter K118011.
K861172: BBa_P0451, a cI generator with RBS and terminator, was embedded after promoter BBa_K118011 activated by CRP.
K861169: K861172 and I763007, a cI repressed RFP generator, were assembled .
K861174: BBa_K137115, constitutively expressing cI generator with promoter, RBS and terminator, was assembled to I763007.
All new composite parts mentioned above were transformed to competent cells of Escherichia coli str. DH5a. All positive clones are validated using PCR, restriction enzyme digestion and DNA sequencing.
Cell culture fluorescence measurement
Minimal medium with different concentration of glucose(1mM, 4mM, 10 mM , 20 mM , 50 mM ,100 mM) were transferred into a 96-well plate, 200 μL for each well. Then each well was inoculated with 2 μL of seed liquor which was activated overnight in M9 minimal medium with 50mM glucose at 37℃. The wells without inoculation were regarded as blank controls to revise the results. Under each condition, three parallel samples were setted. The plate was incubated at 37℃, 150rpm. Cell culture fluorescence was recorded on a SpectraMax M2 plate reader (Molecular Devices). Excitation at 584 nm and emission at 607 nm were used. All fluorescence was normalized with cell density by measuring the absorbance at 600 nm.
Capturing fluorescent image
Cell morphology was observed through fluorescence microscope, and the image of bacteria with of each glucose concentration were captured. To know more about these images, please click on Here.
Fluorescent analysis of cyto-imaging
A program named FANCY was designed to recognize single cell and calculate the fluorescence strength according to the images. For more information, please click Here.
Results
Purified plasmids constructed before were digested with XbaI and PstI for confirmation. The agarose gel electrophoresis showed that the lengths were correct. At last, the plasmids were sent for sequencing. Results showed no mutation.
In the cell culture fluorescence measurement experiment, fluorescence of BBa_K861173 decreased coordinating with glucose concentration, while BBa_K861169 was reverse.The fluorescence of BBa_K861174 was too low to record, so we do not show it here. All of the results coincided with expected results indicating that we have successfully constructed the promoter which was activated by high concentration of glucose.
Fluorescent images indicated that all cells were growing normally, because the size and morphology were both the same as cells in LB medium. The fluorescence of the cells in the images show the same discipline with results from the fluorescence measurement experiments.
The results of FANCY are showed as bellow, which conforms well the results that showed above.
Discussion
All results of the three experiments indicate the device works as expected. Next, RFP will be replaced with genes of cellulose synthesis. So the exceeded glucose can be transformed into cellulose.
Although the indirect regulation pathway was tested effective,it works through a intermediate product, protein cI. This determines that the device will be less sensitive to glucose than a direct regulation pathway without intermediate.
Direct Regulatory Promoter Design
We designed promoter Pcar based on promoter BBa_J23119, inserting CRP-binding site to overlap on six base pairs with promoter -10 region. Since steric hindrance of CRP dimer blocks the function of -10 region, gene downstream will be repressed when glucose concentration is low. That is, most CRP appears in DNA-binding configuration. The repressive effect is undermined when glucose concentration increases. Accordingly, we changed CRP from an activator to a repressor, simplifying the device with potential advantages of higher sensibility and efficiency. As experimental results show, promoter Pcar works as we expect.
Methods
Design of the promoter Pcar which is activated by glucose
Promoter Pcar , glucose biosensor plasmid, is derived from constitutive promoter (BBa_J23119) by adding a CRP binding site upstream the promoter which has several base pairs overlapping with polymerase binding site. The sequence was synthesized with restriction enzyme cutting site for EcoRI and XbaI at the 5' terminal and SpeI at 3' terminal. The sequence of promoter Pcar has cohesive terminus at both ends, so it is very convenient for us to construct the plasmid for functional detection.The sequence of Pcar is as followed:
Construction of plasmid for direct regulation pathway
In this experiment, RFP reported the function of the indirect regulation pathway.
K861179: BBa_I13507, an mRFP generator with RBS and terminator was embedded downstream the constitutive promoter BBa_J23119.
K861176: BBa_I13507 was embedded downstream the artificial promoter Pcar.
K861178: a constitutive expressed CRP(J23116+K861161) was assembled with K861176.
All new composite parts mentioned above were transformed to competent cells of Escherichia coli str. DH5a. All positive clones are validated using PCR, restriction enzyme digestion and DNA sequencing.
Functional detection
The same methods with that of the indirect regulation pathway were used to confirm that the promoter worked as expected. For details, please click Here.
Results
Construction of the plasmid for functional detection
The sizes of the Promoter Pcar and J23119 were less than 100 bp and proved to be correct by the agarose gel electrophoresis . Restriction Digestion of the plasmid BBa_I13507 only have one lad on the agarose gel, it told that the plasmid was digested well. After transformation, competent cells were cultured on agar plate with 50 μg/L of ampicillin. Both red and white bacterial colonies emerged on one plate. The red ones were the correct clones revealing promoter embedded successfully, while the white ones were negative . The red clones were picked out and cultured in LB medium for plasmid extraction. Purified plasmids were digested with XbaI and PstI for confirmation. The bands of 2000bp and 1000bp showed that the promoter had been embedded successfully. At last, the plasmids we acquired were sent for sequencing, results show no mutation exist.
Cell culture fluorescence measurement.
The correct clones were cultured in 96-well plate at 37℃ for 24 hours,then the fluorescence and absorbance at 600 nm were recorded on a SpectraMax M2 plate reader. All fluorescence was normalized with absorbance at 600 nm.The results represented the fluorescence of every cell.
The fluorescence of K861179 was about 10000 Relative Light Units. It did not vary with concentrations of glucose. However we found a positive correlation between the fluorescence of K861176 and concentration of glucose. At a glucose concentration lower than 4mM, the fluorescence was very low, but at high concentration of glucose like 100mM, the fluorescence was much less than that of K861179.
Capturing of the fluorescent image
Fluorescent images indicated that all the cells were growing normally, because the size and morphology were both the same with cells in LB medium.the fluorescence of the cells in the images show the same discipline with results from the fluorescence measurement experiments. Fluorescence of K861179 was very strong but it didn't change with the glucose concentration. On the contrary, fluorescence of K861176 was relatively weak but increased with concentration of glucose.
Fluorescent analysis of cyto-imaging
The results of FANCY is showed as bellow, single cell was recognized from fluorescence images and fluorescence intensity was caculated.In the table, datas show that RFP expression was activated in high glucose concentration, which conforms well with results above.For more information about FANCY,click here.
Discussion
The promoter Pcar is a promoter designed for the Eschaerichia coli which is derived from a constitutive promoter BBa_J23119. Pcar includes the CRP-binding site and the RNA polymerase-binding site which overlap several base pairs. Therefore, because of the steric hindrance between CRP and RNA polymerase, gene downstream of the promoter will be repressed at high concentration of CRP. In the cells, low glucose concentration results in increasing activity by adenylate cyclase. cAMP binds to the cAMP receptor protein, which, in its bound form, is able to bind tightly to the specific DNA site in the promoter and repress the gene downstream. On the contrary, high glucose concentration will result in the expression of the promoter.
Fatty Acid Degradation Device
Purpose
To help people lose weight without the need of food restriction, we designed a genetically modified E.coli that can sense and degrade excessive fatty acids intake by the host. We hope that, together with other two devices we designed, we can introduce our E.coslim as resident in intestine to consume the excessive calories intake by the host and regulate intestinal microbiota.
Outline
Genes that are responsible for degradation and transportation of fatty acids (FAs) from E.coli K12 and from
Salmonella enterica LT2 were cloned. Also, a promoter that can that can be regulated soley by fatty acids was also
designed. By placing those fatty acid degradation genes downstream of the artificially designed promoter PfadR (BBa_K861060) that can sense
the concentration of FAs, we hope to create a device that to degrade FAs only when the concentration of FAs is high.
Long chain fatty acids are firstly being imported by the transmembrane protein FadL. After FAs get into cells, a CoA will be added by inner membrane-associated FadD (acyl-CoA synthase). β-oxidation is initiated by FadE(acyl-CoA dehydrogenase), which will convert acyl-CoA into enoyl-CoA. The following cycles of hydration, oxidation, and thiolytic cleavage are carried out by tetrameric complex consisting of two FadA and two FadB proteins or two FadI and two FadJ in anaerobic condition. FadR is a transcriptional regulator that, when not binds to acyl-CoA, can either serve as an activator for fatty acid synthesis gene like FabA, FabB and etc. or a repressor for fatty acid degradation gene like FadA, FadB, FadD FadE, FadL, FadI, FadJ and etc. After long chain fatty acids are converted to fatty acyl- CoA by FadD, it can bind to FadR. The binding will alter the conformation of FadR, making FadR unable to bind to the DNA sequence it recognizes to fulfill its function. Therefore, FadR can no longer activate or repress the transcription of genes downstream FadR binding sites. However, to our knowledge, there is no promoter exists in nature that can respond solely to FadR since those promoters are often regulated by glucose concentration or oxidative stress and many other factors. In our design, FadL, FadD, FadE, FadA, FadB FadI, FadJ and FadA from Escherichia coli K12, and FadA, FadB and FadE from Salmonella enterica LT2 are placed downstream a synthetic promoter PfadR to make them under the sole regulation of fatty acids concentration.
Progress
Cloning of the gene
First, the genome of Escherichia coli K12 str. DH5ɑ and Salmonella enterica LT2 (symbolized as S-) were got and amplified in PCR using primers for each gene. The sequences of the primers used are as bellow (5’---3’). FadR Forward: GGAATTCTCTAGAATGGTCATTAAGGCGCAAAG Reverse: GACTAGTCTTATCGCCCCTGAATGGCTAAATC FadA Forward: GGAATTCTCTAGAATGGAACAGGTTGTCATTGTCG Reverse: GACTAGTTTAAACCCGCTCAAACACCGT FadB Forward: GGAATTCTCTAGAATGCTTTACAAAGGCGACACC Reverse: GACTAGTTTAAGCCGTTTTCAGGTCGCC FadD Forward: GGAATTC TCTAGATTGAAGAAGGTTTGGCTTAACCG Reverse: GACTAGTTCAGGCTTTATTGTCCACTTTGC FadE Forward: GGAATTC TCTAGAATGATGATTTTGAGTATTCTCG Reverse: GACTAGTTTACGCGGCTTCAACTTTCCG FadL Forward: GGAATTC TCTAGAATGAGCCAGAAAACCCTG Reverse: GACTAGTTAGAACGCGTAGTTAAAGTTAG FadI Forward: GGAATTC TCTAGA ATGGGTCAGGTTTTACC Reverse: GACTAGTTTATTCCGCCTCCAGAACCA FadJ Forward: GGAATTCTCTAGAATGGAAATGACATCAGC Reverse: GACTAGTTTATTGCAGGTCAGTTGCAGTTG S-FadA Forward: GGAATTCTCTAGAATGGTCATTAAGGCGCAAAG Reverse: GACTAGTCTTATCGCCCCTGAATGGCTAAATC S-FadB Forward: GGAATTCTCTAGAATGCTTTATAAAGGCGACACC Reverse: GACTAGTTAAGCCGTTTTCAGAGAACC S-FadE Forward: GGAATTCTCTAGAATGATGATTTTGAGTATTATCG Reverse: GACTAGTTATGCGGCTTCGACTTTACGC
Design of the Promoter PfadR Repressed by Fatty Acids
Promoter PfadR, is derived from BBa_J23110. Specifically, FadR binding site of FadL gene is placed overlapping with the last 3 base pairs of BBa_J23110 The sequence was synthesized with restriction sites for EcoRI and XbaI at the 5' terminal and SpeI at 3' terminal. We use overlapping PCR to get the double strand DNA. The sequence design of PfadR is as followed: Forward: GGAATTCTCTAGATTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGCTGGTCCGACCT Reverse： GACTAGTTCTTAGAAATCAGACCAGTGGCGAGAGTATAGGTCGGACCAGCTAGCATTGT
Construction of Biobricks
Fatty acid degradation project is divided into two parts: Promoter, and gene function To discover the optimal combination of those fatty acid genes, we: 1. PCR to clone those genes in E.coli K12 and Salmonella enterica LT2 2. Restriction digest and ligate those gene into pSB1C3 3. Restriction digest and ligate those gene with RBS(B0030) 4. RBS-FadA, RBS-FadI, and RBS-S- FadA is ligated with both BBa_R0011 promoter and our PfadR RBS-FadR, RBS-FadB, RBS-FadJ, RBS-FadE, RBS-FadD, RBS-FadL, RBS-S-FadB, and RBS-S-FadE are ligated with B0034 5.PROMOTER-RBS-FadA is ligated with RBS-FadB-Terminator, PROMOTER-RBS-FadI is ligated with RBS-FadJ-Terminator and PROMOTER-RBS-S-FadA is ligated with RBS-S-FadB-Terminator. RBS-FadE-Terminator, RBS-FadD-Terminator, RBS-FadL-Terminator, and RBS-S-FadE-Terminator, are ligated with BBa_R0011 promoter, PfadR and various constitutive promoters. For Promoter PfadR 1. promoter PfadR was synthesized using overlapping PCR 2. RFP reporter was ligated downstream the promoter and ligted into pSB6A1 3. J23116+ RBS+ FadR+ Terminator was ligated to PfadR+ RFP in pSB6A1
Experimental Procedure
We used cupric acetate-pyridine as a color developing reagent to determine fatty acid consumption of genetically modified bacteria. We had modified existing methods to extract free fatty acid in M9 medium. Also, we used IPTG induced promoter BBa_R0011 to see the expression of those proteins and extract proteins from cells. For more details, please see Protocol page.
Results
Characterization of each gene
In this experiment, we wanted to test whether the ability of degrading fatty acids of our genetically modified bacteria was enhanced as expected by transforming plasmids constitutively expressing related genes in the beta oxidation pathway. The effects of the genes we have tested is listed in the following chart I. The ability was reflected by the change of the concentration of the fatty acids in the medium. It was measured by cupric-acetate soap reaction as described Protocols section.(此处可给一个链接) Each time we inoculated 50mg bacteria into 30ml M9 medium using fatty acid as sole carbon source, collecting the sample at the time as shown in the picture. Then the analysis of the free fatty acids was performed.
图 As our data suggests, PfadR- FadL and J23114- FadL can degrade the fatty acids better than the control. They are also faster than the control, bacteria express protein irrelevant to fatty acid degradaton of similar size.Others are not that obvious. The column figure is present as below. The result of the analysis of the variance is present in chart II.
Colonization
Purpose
One big challenge of probiotics is their survival in intestine. We respond to this challenge by expressing gene adrA responsible for manufacturing the second messager c-di-GMP, a magic molecule that leads to inhibition of motility and increase of adhesion and division of E.coli.
Outline
AdrA protein can convert GTP into c-di-GMP, a magic second massager that, besides promoting the production of cellulose(for more details, see our celluose synthesis device), can reduce the expression of flagella and acute virulence gene simultaneously. In the same time, c-di-GMP can facilitate the synthesis of various adhesins and exopolysaccharides and can promote the proteolysis of replication inhibitors. As a result, AdrA makes cells become adhesive and promote the formation of biofilm, making the bacteria gain advantages to survive in the hostile environment of intestine.
Experimental Procedure
We tested the function of AdrA gene by plate assay. Formore details, please visit our protocol page.
Results
As can be seen from the plate. Clone with AdrA constitutively expressed using BBa_J23107 is much more smaller compared to the control that only contain the plasmids of RBS, though the amount of bacteria is similar. This result showed that the AdrA was successfully expressed, elevating c-di-GMP level, leading to the increase expression of adhesins and inhibition of motility.
