Wet Lab Results

Calibration

Calibration of OD with colony forming units

Chemoinduction

IPTG Growth

Autoinduction

How GFP expression from CRP-GFP construct varies with different media

Thermoinduction

Thermoinducible promoter part 1

Thermoinducible promoter part 2

Module 2

Colanic acid results

Major results are shown in this section; there's a brief description of each and you can click through to find more detail on them. For minor results, please refer to our Notebook.

Module 3

Restriction Enzyme testing in Dam+ve and Dam-ve Strains

An experiment investigating the effects of methylation on the amount of cleavage by varying concentrations of restriction enzymes.

Others

Calibration

OD calibration

OD calibration

• To make a calibration curve linking our O.D. 600 to colony forming units.

23rd September

2nd October

Module 1

Module 2

pH and GFP

How cell lysis varies with pH (using GFP as marker). Chemically induced encapsulation 18-09

Module 3

Genome Deletion

Restriction Enzyme testing in Dam+ve and Dam-ve Strains

An experiment investigating the effects of methylation on the amount of cleavage by varying concentrations of restriction enzymes.

Temporal Control

Chemical induction

Cell growth for different IPTG concentrations

Testing if IPTG toxicity affects cell growth, and consequently, production of protein of interest.
Rationale: IPTG has been shown in the past to have toxic effects on cell growth if administered in large quantities [http://openwetware.org/wiki/IGEM:IMPERIAL/2009/M1/Modelling/Analysis/literatureIPTG (literature review IPTG)]. However, production of the protein of interest will be induced with IPTG and from the Modelling results, output yield of protein of interest will increase with concentration of IPTG, provided that the levels are non-toxic. The results we present here are a study on how IPTG exerts its effects on cellular growth rate, in order to determine if the concentrations we are using have negative effects on the cultures, and consequently, how this will affect output levels of drug of interest.

Lac-RFP tests

Prelim results on 13th October

Autoinduction

Diauxic growth

8th September

10th September

Growth in the presence of Glucose only

Testing the effects of glucose concentration on cell growth: This experiment will be linked to diauxic growth.
Rationale: A cell culture in the presence of glucose grows exponentially. Once it uses up its resource, it stops growing and the population density saturates.
Raw data 06/10 Media:II09_rawdata.xls

Growth in the presence of Glucose + 2ndary carbon sources

Testing the effects of different secondary carbon sources on cell growth with a fixed initial glucose concentration. Rationale: A cell culture in the presence of glucose grows exponentially. Once it uses up its resource, it stops growing and the population density saturates. However, in the presence of a secondary carbon source, the cells enter a second exponential phase where they grow and saturate once again when the secondary source has been used up.
NOTE: Same raw data file as above!

Growth in the presence of Glucose + Xylose

Testing the effects of different xylose concentrations on cell growth, for a fixed initial glucose concentration: This experiment will be linked to diauxic growth.
Rationale: As above

CRP promoter

Review on CRP promoter results

Prelim test-Growth on glucose and xylose and effect on CRP promoter 07-10
A simple test of all xylose and all glucose to test if GFP is generated. Found out that GFP levels are about the same and quite low for both xylose and glucose (ie both do not express)

CRP promoter and varying glucose 09-10

Growth on different media and effect on CRP promoter 10-10

Supposing that Secondary carbon sources repress the CRP promoter, we proceeded to test which media gives the best CRP output. It appears to be 10% Casamino acid.
download raw data

Prelim test on GFP and RFP production from construct CRP-GFP-Lac-RFP

CRP promoter appears to be constitutively on, whether Glucose or Xylose is present.

Most likely because 0.05% glucose is unable to repress CRP promoter.

CRP promoter and varying glucose 13-10

CRP promoter and varying glucose 14-10

Download raw data here

Thermoinduction

Harvard+GFP results 6-10

Testing the effects of temperature on fluorescence output.
Rationale: Genome deletion is repressed at low temperatures, and triggered when the temperature rises. Here, we are testing the p-lambda promoter by attaching GFP to it, in order to show how the fluorescence output varies when the temperature rises. raw data
6th October
23rd September