From 2011.igem.org

August 10

Today the attached procedure outlined for synthesizing quantum dots biologically was followed for each of the bacteria cultures made on August 8. However, when samples were prepared using this protocol, certain variables were left out. This decision made these samples into controls. As such, they were used to determine whether quantum dots had actually been synthesized last week.

Notice, in the list of samples prepared below certain samples were made excluding one of the following variables: cadmium chloride solution, sodium sulfide or bacteria.

1. DH5alpha, Cadmium Chloride and Sodium Sulfide
2. CDS7, Cadmium Chloride and Sodium Sulfide
3. 127, Cadmium Chloride and Sodium Sulfide
4. CDS7 and Sodium Sulfide
5. CDS7 and Cadmium Chloride
6. Cadmium Chloride and Sodium Sulfide

It was unclear whether quantum dots had been synthesized because each supernatant of each controls fluoresced. It was found that LB broth is fluorescent. Because LB has this quality and is present in all of our samples, the presence of quantum dots can not be confirmed using fluorescent spectrometry at this time. The quantum dots supposedly produced last week may not be present and the fluorescence of the sample attributable to the LB broth alone.

August 8

Today was used in preparation for experiments planned for the following days.

In order to verify the production of quantum dots accomplished last week, bacteria containing the following parts were cultured separately:

1. CDS7
2. Rel Light Promoter and GFP (labeled as 127, the last three digits of its part identification number)
3. DH5alpha

These bacteria were cultured in LB prepared today. 720 ml of LB was made all total.

August 2

The procedure for August 1 was repeated, but less reliable results were obtained.

August 1

Task 1: Today we developed a protocol to be completed tomorrow outlining the procedure to synthesize quantum dots. The protocol was adopted from the journal article "Biosynthesis and Characterization of CdS Quantum Dots in Genetically Engineered E. Coli."

Note, instead of using the pET - 28b vector, which requires IPTG as a promoter, our protocol calls for pMA vector, requiring no promoter.

The CdCl₂ and Na2S[9H₂O] solutions necessary for our protocol were prepared today.

Task 2: The procedure completed last Thursday, July 28, will be repeated tomorrow, Aug 2. Today, the bacteria containing the following parts were inoculated.

1. pUC19
2. pUC19
3. GFP and arabinose
4. GFP alone
5. BL alone
6. BL with GFP
7. BL with GFP
8. CD7

July 28

Minicultures of cultures made the evening before were prepared with the following components:

• 3.5 ml of LB broth
• 4 microliters of Ampicillin
• 4 microliters of Arabinose when required
• 0.5 ml of appropriate culture

Using the components listed the following minicultures were prepared. Their labels refer to how the cultures would be incubated, either in the presence of blue light from the LED box or in the absence of light.

1. 470 nm Blue Light Promoter (BLP) and Green Fluorescent Protein (GFP)
2. unused sample
3. 470 nm pUC19
4. Dark BLP and GFP
5. unused sample
6. Dark pGLO without Arabinose
7. Dark pGLO with Arabinose
8. Dark pUC19

Cultures 1 and 3 were set to incubate in the LED box constructed on July 27. The remaining cultures were set to incubate in a hot water bath in the dark. Phosphorescent readings were taken at timestamps 30min, 60, 120 and 180 for each culture. Each culture was sampled in triplicate on a 96 well plate using LB broth as the control.

July 27

A goal of our project is to use LED lights to signal bacteria into synthesizing quantum dots. Today, the LED box was constructed. Two bread boards were lined with about 50 LEDs (Light Emitting Diodes), resistors (330 Ohms), and a 9 Volt battery. This plate was fastened to the ceiling of a foil lined box. A switch was also soldered into the circuit. The box is large enough to hold multiple minicultures, preps in which cells will be incubated. The blue light from the LEDs used will signal the blue light sensors transformed into the bacteria which will in turn initiate the synthesis of quantum dots.

July 25

Gels of agarose concentrations 1.5%, 2% and 3% were loaded with prepped digest samples from July 21. The gels were loaded as follows. Single and double refer to the type of digest. (Two 1.5% gels were used.) The gels were analyzed during the next lab session.

July 21

Made minipreps of pUC19, K238015, and K238013. The following were calculated from the samples: Plasmid concentration in each sample; Volume of sample required for 1 microgram of DNA. This information determined how the plasmids would be prepared for the single and double digests.

Sample	Concentration (microgram/milliliter)	Volume Required for 1 microgram of DNA
K238015 (4)	0.2	5.0
K238013 (8)	1.7	.59
pUC19 (15)	3.6	.28

Unfortunately the concentration of DNA in each of the samples was found to be low. The digest solutions prepared contained buffer, enzyme and DNA but no water, in order to compensate. These samples were properly incubated, following the digest preparation procedure and then stored.

Electrophoresis gels were prepared for use in analyzing the digest preps the following day.