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Project

Lab Notebook / Lab Journal

Weeks 1 and 2 (June 1st - June 12th)

After many months of looking for a lab where we could work and after planning our project, we arrived to our lab with the Wetlab assesor and began sterilizing some material and locating many things we are going to use.

Also, our guests from the UNAH arrived and we were pleased to teach them some Molecular Biology and Protein’s Physicochemistry principles, as well as how to use scientific software and how to navigate in iGEM’s databases. They are staying in Mexico for 4 weeks.

Weeks 3 and 4 (June 15th - June 26th)

We went to Cuernavaca in order to speak with many researchers about our projects (ours and the one of our guests); we went to the Biotechnology Institute, the Center for Genomic Sciences and the Physics Institute.

We began some Wetlab practices with our UNAH guests and we inoculated the E. coli strains that we are going to use in our project. These strains are:

EnvZ -/- An E. coli strain with a deleterious mutation in the EnvZ receptor, which senses osmotic changes in the media and is part of the EnvZ/OmpR two component system.

SHuffle A strain with a cytoplasmic oxidative environment capable of making disulphide bonds in many peptides (which a WT strain is no able to form).

Rosettagami A strain used to synthesize many folded proteins which cannot be right folded by a WT strain.

DH5α Typical WT strain used for molecular biology steps like cloning vectors and amplifying plasmids. This strain methylates its DNA.

JM110 WT strain which doesn’t methylate its DNA, it is used mainly to obtain plasmids for digestion reactions.

All strains were cultured in LB medium

After we amplified our cultures, we were able to make competent cells with CaCl2 and we tried to transform EnvZ-/- and DH5α with our main reporter gene, RFP under OmpR promoter, but got only a single colony in DH5α strain, so we are going to make competent cells again.

Weeks 5 and 6 (June 29th - July 10th)

We followed DH5α, EnvZ-/-, SHuffle and Rosettagami’s growing rates by measuring absorbance at 600nm. They were cultured in complete LB medium.

We made competent cells again with CaCl2 and were able to transform a constitutive GFP and YFP to confirm that our cells were competent. After this confirmation, we transformed our reporter RFP from part BBa_M30011 in DH5α and EnvZ-/- strains.

Weeks 7 and 8 (July 13th - July 24th)

We prepared the solutions needed for a homemade Miniprep and got much more BBa_M30011 plasmid, so we transformed more EnvZ-/- strain with it and amplified both DH5α and EnvZ-/- cultures containing the reporter gene; we kept amplifying cultures with constitutive GFP and YFP in order to use those plasmids to test other techniques.

We measured the LB medium’s osmolarity, since it is important for our project to control it; our LB medium’s osmolarity was 420 mOsm/L so we are going to dilute it to reach an osmolarity of 300mOsm/L, which is the osmolarity of intersticial fluid. We planned the curve we are going to measure in an ELISA plate in order to see how this reporter gene (“OmpR controlled mRFP”) behaves in different conditions of glucose concentration and medium osmolarity.

Weeks 9 and 10 (July 27th - August 7th)

We measured our reporter gene’s expression by measuring it’s absorbance. RFP’s excitation wave length is 560nm and it’s emission wavelength is 612nm. The curve was made for 5 hours and measurements were made every half an hour.

Weeks 11 and 12 (August 10th - August 21st)

Our DNA fragments and oligos finally arrived along with the NEB’s Gibson Assembly Mix so we could finally assemble both of our parts, the Trg-EnvZ chimera and Insulin Lispro, both of them are built under a constitutive promoter to test wether or not they are expressed in our strains.

Weeks 13 and 14 (August 24th - September 4th)

We confirmed the assembly of our parts and we transformed them in our strains to obtain a large quantity of plasmid; we also assembled the parts in PSB1C3 backbone to deliver it to iGEM Headquarters while we test their expression by Western Blot (in the case of insulin) and by stimuli response (in the case of the chimera). We assembled our constructs and after transformation and selection of three clones on the plates, minipreps were performed and subsequent digestión with Xbal was done.

Tar-EnvZ chimera assembly:

Expected bands after Xbal digestion

Obtained bands after Xbal digestion

Obtained band after Xbal digestion

Parts

Parts were designed by Alejandro Rodríguez and Lizbeth Bolaños mainly using the Serial Cloner software.

For both parts, the sequence was obtained by back-translating the aminoacid sequences of interest into nucleotides; then restriction sites like EcoRI, NotI, XbaI (among others) were removed maintaining the open reading frame and finally the nucleotide sequences were codon-optimized for E. coli.

Trg-EnvZ chimera

The Chimera Trg-EnvZ is part of a two component system and it constitutes a chimeric protein composed of the extracellular and transmembrane domains of chemoreceptor Trg and the cytosolic kinase domain of osmosensor EnvZ . Trg is activated by periplasmic binding proteins, and transduces the message to EnvZ to phosphorylate OmpR, a transcription factor which binds to the OmpC promoter driving transcription of whichever gene that is downstream.

Insulin lispro

Insulin lispro is a rapid-acting insulin analog capable of lowering blood glucose levels; it differs from native insulin in that it has an inversion between proline al position B28 and lysine al position B29. This part has a constitutive promoter and an excretion signal from TAT system at its N-terminus

BioBricks.

BBa_K1731001

Insulin lispro is a human insulin analog that dissociates more rapidly than human regular insulin after subcutaneous injection, resulting in higher insulin levels at an earlier point in time and a shorter duration of action1. We designed a sequence of constitutive expression for insulin lispro. Our aim is to express lispro as proinsulin containing peptide C and with a TAT sequence in its N terminus. This construct is expected to be expressed in Rossetagami and Shuffle, in these two strains where eukaryotic protein expression is enhanced we expect to have a fully former insulin with disulfide bonds.

In a future we would replace the promoter of this construct for an OmpC promoter so that its expression can be modulated by EnvZ activation.

1. HoweyDC, BowsherRR, BrunelleRL, Woodworth JR: [Lys(B28), Pro(B29)]- human insulin: a rapidly absorbed analogue of human insulin. Diabetes 43:396-402,1994

Results:

Insulin Expression in Rossetagami (R) and SHuffle strain. A western blot was performed with transformed strains with Lispro construct and controls from both strains. Our biobrick is expressed in the two strains, this result only confirms the protein is expressed but further analysis needs to be done in order to confirm fully formed insulin with disulfide bonds.

BBa_K1731000

Two component systems is the major signal transduction mechanism in bacteria, several receptor can sense some compounds in the media to response with chemotaxis and metabolic changes. There are several periplasmic binding proteins that are the firs sensor to a broad range of molecules, one of our interest is Glucose Galactose Binding Protein (GGBP)(Dwyer and Hellinga 2004). Glucose is sensed by bacteria through the GGBP which has been proposed as a glucose sensor (Hsieh et al. 2004). Trg-EnvZ was initially created in 1994, in order to measure the carbohydrates concentration and using the EnvZ intracellular with a reporter, Trg chemoreceptor triggers the chemotaxis of the bacteria while the EnvZ is an osmoregulation which actives the transcription factor OmpR. This chimera allows an easy way to measure glucose concentration using a reporter for EnvZ(Tolosa and Rao 2006).

Dwyer M a., Hellinga HW. Periplasmic binding proteins: A versatile superfamily for protein engineering. Curr. Opin. Struct. Biol. 2004;14(4):495–504.

Hsieh H V., Pfeiffer Z a., Amiss TJ, Sherman DB, Pitner JB. Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein. Biosens. Bioelectron. 2004;19(7):653–60.

Tolosa L, Rao G. The Glucose Binding Protein as Glucose Sensor: Protein engineering for low-cost optical sensing of glucose. Top. Fluoresc. Spectrosc. Vol. 11 Glucose Sens. 2006;11:323–31.

REFERENCES:

• Redondo M. et al. Genetics of Type 1ª Diabetes. N Engl J Med 2009; 360:1646-1654

• Impact of Fat, Protein, and Glycemic Index on Postprandial Glucose Control in Type 1 Diabetes: Implications for Intensive Diabetes Management in the Continuous Glucose Monitoring EraDiabetes Care June 1, 2015 38:1008-1015

• American Diabetes Association. Standards of Medical Care in Diabetes-2015: Abridged for Primary Care Providers. Clinical Diabetes. 2015. 33(2)

• [Guideline] Diagnosis and classification of diabetes mellitus. Diabetes Care. 2010 Jan. 33 Suppl 1:S62-9.

• [Guideline] American Diabetes Association. Standards of medical care in diabetes. 2012. Diabetes Care. 2012 Jan. 35 Suppl 1:S11-63

• Frontiers in Diabetes. Technological Advances in the Treatment of type 1 diabetes; vol. 24. Germany 2014

• Boron, W. F., Boulpaep, E. L. Medical Physiology, 2nd Ed., Elsevier – Saunders. New York, 2012.

• Berne & Levy, Physiology, 6th Ed., Koeppen, B. M., Stanton, B. A. (Eds.),

• Mosby, Philadelphia, 2008.

• Guyton and Hall. Textbook of Medical Physiology. 12nd Ed., Elsevier 2011.

• Romesh khardori. (2015). Type 2 Diabetes Mellitus. Retrieved 13 September, 2015, from http://emedicine.medscape.com/article/117853-overview#a2

• Whoint. (2015). Whoint. Retrieved 13 September, 2015, from http://www.who.int/mediacentre/factsheets/fs312/en/