Team:IISc Bangalore/Experiments

Team IISc iGem

Experiments

BBa_I746908

characterization

E. coli BL21 (DE3) was transformed with BBa_I746908 and a single colony was inoculated into LB broth (with the appropriate concentration of antibiotic) primary that was grown overnight (37 celcius, 180 rpm) and then 1% of the primary was inoculated into 8 x 250mL conical flasks, 2 each with the following:
1. 100mL LB broth (blank)
2. 100mL LB broth + 0.2% glucose
3. 100mL LB broth + 0.2% arabinose
4. 100mL LB broth + 0.2% arabinose + 0.2% glucose
0.5mL samples were collected every hour in microfuge tubes and stored at 4C and the flasks were incubated at 37C, 180rpm. OD600 and fluorescence values of the samples were recorded.

BBa_K1352000

characterization

1. Four 25 ml of LB media in conical flasks were inoculated from an overnight primary culture of E. coli BL21 (DE3) transformed with appropriate part (BBa_K1072000 for lux part and BBa_R0040 for negative control) in pSB1C3 (1 % inoculation, 37 degree celcius, 180 RPM).
The cultures were grown in an orbit shaker incubator at 37 degree celcius, 180 RPM.
200 uL aliquots were taken from each conical flask every 2 hours and stored at 4 degree celcius.
GFPmut3 fluorescence (Ex: 501 nm, Em: 511 nm) and OD600 of the samples were recorded on a plate reader (Tecan M2000 InfinitePro).

For results of these experiments, please refer to Results page.

Overview

With the advent of rDNA technology in the late 1970s, medicine, agriculture and several other areas underwent a quantum leap and from that point, progress only hastened, from one only one recombinant pharmaceutical approved for human use (insulin) in 1982 to one hundred and fifty-one FDA approved protein based recombinant pharmaceuticals by 2009[1]. Despite being in high demand (due to the fact that most recombinant products produced on an industrial scale are therapies for chronic diseases like cancer and diabetes), recombinant products are expensive due to several factors like long and expensive development time, high failure rate (~80%) of the products developed, manufacturing costs requiring expensive technologies and processes (bioreactors, column chromatography, sterile conditions, etc) and the involvement of skilled labor on both the manufacturing and the healthcare provider’s side[2]. Treatments with these pharmaceuticals can cost from around 10,000 to 100,000 € per year for a single patient[2]. As scientists and engineers, it seems obvious that our contribution can be most easily and effectively be made at the level of manufacturing costs; to try to bring down the cost of these life-saving products.
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Bioreactor design

We realized that manufacturing units use expensive and energy expensive machinery [3]. Hence, we engineered our bacteria in such a way that it could do most of the work done by these monstrous machines so that we no longer have to buy them and pay their bills. To increase the yield of the product, we came up with the idea of separating cell growth and target protein production so that when the cells have grown up to a high enough cell density, they shuttle all their energy towards the required protein production [4]. Based on the above ideas, we came up with the design of our bioreactor.

We realized that we would not be able to work on the whole idea this summer so, we focussed on two modules of our design: the Protein production module and the auto-aggregation module leaving the arresting of cell growth for future iGEM teams as a seed for their ideas.
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Protein production

Induction synthesis of protein of interest is another important step. Microorganisms in bioreactors are induced at the OD, at which the protein production is maximum. [5] Because of this, factories install sophisticated machinery to keep track of the OD value of the culture so as to know when to induce the production. We wanted our bacteria to know when to start protein production and start it by itself. Hence, we used the lux quorum sensing system to sense this cell density. [6] We have expressed the gene for our protein of interest under the luxI gene so that protein of interest is produced in significant quantities only when the quorum is achieved.(Figure 2) We also wish to tune the quorum sensing system by tweaking with the strength of the promoters involved in quorum sensing.
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Auto-aggregation

To achieve cellular aggregation, we use the famous auto-transporter protein, Antigen43 (Ag43). [7] It has shown to cause auto-aggregation of cells when overexpressed. (Figure 3) We plan to exploit this property of Ag43 to substitute centrifugation. We acknowledge previous iGEM teams who have worked on Ag43 (Hokkaido University, 2012; Aberdeen Scotland, 2014 and some more) as we got the BioBricks for Ag43 readily available. We intend to express Ag43 only when the cells have made the product of interest. Hence, we use the diauxic shift in the media to act as a switch for the expression of Ag43.
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