Team:IISER Tirupati/Experiments
We used DH5a strain of E. Coli for cloning and BL21 (DE3) as chassis/expression vector. sfGFP was used as reporters in our constructs. The plasmids we used were psb1C3 (promoter regions and sfGFP) and psb3K3 (structural elements of lldPRD operon).
The fragments of choice were ordered and were ligated together using SOEing wherever necessary, and the clones were confirmed by colony PCR and insert release. We chose SOEing for our constructs because it provided us with a cost effective option that could give us our library of constructs within a short span of time.
Simultaneously, our constructs were sent for sequencing as we got them.
For our constructs, we chose to use RBS of different strengths, since a library of various promoters was already screened (ETH Zurich). From that library, we chose the best promoter - J23117. The following constructs from our library were successfully cloned:
- Constitutive Promoter - Strong RBS - sfGFP - Terminator
- O1 - J23117 - O2 - Strong RBS - sfGFP - Terminator
- Constitutive Promoter - Weak RBS - sfGFP - Terminator
- O1 - J23117 - O2 - Weak RBS - sfGFP - Terminator
- Constitutive Promoter - Strong RBS - YebF-IL12-Flag-Terminator
- O1 - J23117 - O2 - Strong RBS - YebF-IL12-Flag- Terminator
- Medium Promoter - Strong RBS - lldD - Terminator
Theory
There are many characteristic properties of cancer cells which differentiate them from healthy cells. One among them is the production of lactic acid and thereby, a lactate rich microenvironment [1]. We use this property to helps our CoCa coli to gain specificity in its function.
Cancer cells reprogramme their metabolism to promote growth, proliferation, and survival. One of the methods is by uptaking more glucose. In cancer cells, glucose uptake increases and fermentation of glucose to lactate takes place even in the presence of completely functioning mitochondria and oxygen. This phenomenon is known as the Warburg effect [2].
The amount of respiration and lactate production varies depending on genetic or environmental influences [3]; thus, it will be different for different tumours. Even though aerobic respiration produces less number of ATP compared to mitochondrial respiration, the rate of glucose metabolism is more in that. This higher rate of metabolism and lower production of ATP is an evolutionary adaptation of cancer cells, which gives them an advantage over healthy cells for limited glucose and nutrients [4].
Warburg effect also supports the biosynthetic requirements of cancer cells, helping them with the uncontrolled proliferation [5].
The increased glucose consumption decreases the pH of tumour microenvironment due to lactate secretion, and this helps the cancer cells to spread quickly [6]. Also, the high rate of glycolysis helps the cancer cells to get more glucose than the Tumour Infiltrating Lymphocytes (TIL) and thereby to escape from the body’s immune system [7]. Thus, the Warburg Effect provides an overall benefit that supports tumour microenvironment to help in cell proliferation and survival.
Detection and quantification
Knowing the amount of lactic acid produced by the cancerous and healthy cells would help us tune our constructs. To detect the amount of lactate produced by the cancer cells, we decided to use HPLC (High-Performance Liquid Chromatography).
Since our project is about colon cancer, we decided to use HT-29 (colon cancer cell line) for the detection and quantification of lactate present in the tumour microenvironment, and CRL-1790 (normal colon cell line) was used as a negative control.
References:
- Geoffrey M Cooper. - The cell, a molecular approach; Development and causes of cancer
- Maria V. Liberti and Jason W. Locasale (2016). The Warburg Effect: How Does it Benefit Cancer Cells?
- Crabtree HG. Observations on the carbohydrate metabolism of tumours. Biochemical journal. 1929;23(3):536
- Pfeiffer T, Schuster S, Bonhoeffer S. Cooperation and competition in the evolution of ATP-producing pathways. Science. 2001;292(5516):504–507.
- Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. science. 2009;324(5930):1029–1033.
- Estrella V, et al. Acidity generated by the tumor microenvironment drives local invasion. Cancer research. 2013;73(5):1524–1535.
- Chang C-H, et al. Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression. Cell. 2015
For our final step, we characterised each of our sfGFP constructs after induction with several different concentrations of lactate. The lactate inducible YebF-IL12 construct was also characterised using western blotting. Throughout our assay, we used sfGFP as our reporter instead of IL12 because of the easily quantifiable fluorescence read-out along with its increased in vitro thermal stability, improved folding kinetics and superior resistance against chemical denaturants in comparison to conventional GFPs.
Our constructs have two kinds of regulatory sequences:
- O1PO2: To make our bacteria sensitive to the lactate that is secreted by colon cancer cells, we used this region from the bacterial lactate operon. Along with this, different types of RBS were used to modulate the sensitivity of the operon to varying levels of lactate in the colon cancer microenvironment.
- Constitutive promoter: This promoter was used to measure the levels of our reporter protein in the constructs. These levels then served as controls with which we compared the production levels of O1PO2. Hence, we could determine the difference between the two and check if our fine tuning was working.
The detailed protocol is available on the protocols page. Check out our results page too!
References: Goers, L. , Ainsworth, C. , Goey, C. H., Kontoravdi, C. , Freemont, P. S. and Polizzi, K. M. (2017), Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol. Bioeng., 114: 1290-1300. doi:10.1002/bit.26254
"We play with bacteria and sometimes they play us"
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Email: igemiisertirupati@gmail.com
Address:
Indian Institute of Science Education and Research, Tirupati
C/o Sree Rama Engineering College (Transit Campus),
Rami Reddy Nagar, Karakambadi Road,
Mangalam (P.O.) Tirupati -517507.
Andhra Pradesh, INDIA
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