Team:WHU-China/Project

From 2012.igem.org

(Difference between revisions)
Line 860: Line 860:
Results
Results
</h3></a>
</h3></a>
-
<p><img src="http://partsregistry.org/wiki/images/e/e7/AdrA.jpg" width="449" height="599" hspace="2" vspace="1" border="2" align="top" /></p></br>
+
<p><img src="http://partsregistry.org/wiki/images/e/e7/AdrA.jpg" width="500" height="620" hspace="2" vspace="1" border="2" align="top" /></p></br>
<p align="justify">As can be seen from the plate. Clone with AdrA constitutively expressed using <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J23107">BBa_J23107</a> is much more smaller compared to the control that only contain <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030">the plasmids of RBS</a>. This result showed that the AdrA was successfully expressed leading to the increase expression of adhesins and inhibition of motility.</p>
<p align="justify">As can be seen from the plate. Clone with AdrA constitutively expressed using <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J23107">BBa_J23107</a> is much more smaller compared to the control that only contain <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0030">the plasmids of RBS</a>. This result showed that the AdrA was successfully expressed leading to the increase expression of adhesins and inhibition of motility.</p>

Revision as of 18:58, 26 September 2012

Project Description

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. One commonly accepted theory is 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, total amount of ATP, ADP and AMP 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 downstream promoter PcstA ( BBa_K118011 ) activated by the binding of CRP, and genes of cellulose synthesis 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 supposed, cellulose production will be increased following the elevation of glucose concentration, and vice versa. For more information, click Here .

Direct Regulatory Promoter Design

Although the indirect regulation pathway was tested effective, we went on attempting a more compact and widely useful direct regulation design. Hence we modified a constitutive promoter ( BBa_J23119 ) to CRP repressible ones. We have established a new technical standard for our strategy of repressible promoter design (for more information, click on Standard ), but we shall focus on the design itself now.

We designed promoter Pcar( BBa_K861171 ) 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, genes 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. For more information, please click Here .

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. This result showed that the AdrA was successfully expressed leading to the increase expression of adhesins and inhibition of motility.