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From 2011.igem.org

Contents 1 Monday 2 Tuesday 3 Wednesday 4 Thursday 5 Friday

Monday

Today we laid out the weekly schedule for assembly, as follows

Day 1: grow culture

Day 2: Assemble (all day)

Day 3: grow colonies and check with PCR

Day 4: miniprep

Day 5: sequence results

Day 6: use miniprep

Note: Every step is done each day for different assemblies

We also began developing preliminary ideas for the summer project:

Proposed Idea	Preliminary Thoughts
Bacterial photosynthesis	Up-regulate bacterial chlorophyll, BCHM=gene to increase chlorophyll production
Microbial fuel cells	Bacteria add electrons to Fe+3 and electrodes to create current, Use sacrificial anode (geobacter)
Serotonin sensor	????
Bacterial arithmetic	Use sRNAs to regulate translation to mirror arithmetic operations based on expression rates

We spent the rest of the day researching these ideas to determine their plausibility.

Tuesday

     Jim started the day off with some information regarding geobacter. Jim explained the goal to optimize geobacter’s production of current in the presence of iron. The proteins responsible in geobacter are called cytochromes. In 2010, Missouri University of Science and Technology created a construct containing some of the cytochromes, so Jim proposed that we look into their part and see if we can optimize it further. The problem with working with Geobacter is its slow growth time, which makes growing cultures difficult. Thus, we determined that this project idea would not be suitable for a summer venture.

     Another idea was proposed today regarding radiation sensing and lambda phage repressors. This idea was proposed by Penn State’s 2007 team, but was never really brought to practice. We proposed a device that contained different strains of bacteria that correspond to different amounts of radiation exposure. Each strain would produce a pigment when exposed to a certain threshold of radiation. After receiving unanimous acceptance, we decided to further develop this radiation sensor. We spent the rest of the day researching the lambda phage system and mechanisms involved in radiation exposure.

     One problem that arose was RecA’s constitutive presence in the cell. RecA promotes homologous recombination, but we do not want recombination between our constructed plasmid and the bacteria’s chromosome. Thus, we needed a mutant of RecA that would still work in the lambda phage system without catalyzing homologous recombination.

Wednesday

     Today, many of the students new to iGEM and lab research at Penn State (Vishal, Alex, Jamie, Kristen, Byron) attended a lecture on handling chemicals and hazardous materials given by Penn State’s Environmental Health and Safety department. After reconvening with the rest of the team, we continued discussing our RecA problem. Jim proposed using a different system (phi80) of repressors used to sense DNA damage, but the RecA mutant used in this system had the same problems of lambda phage. We determined that we would have to make our own mutant of RecA and developed a system to test it, as shown in Figure 1.

     We also looked into our reporter system, which would be activated by the lambda system. We researched the iGEM project submitted by Imperial College in 2010 in order to have a fast reporter system for our biological dosimeter.

Thursday

• Today, we confirmed our radiation sensor circuit, as shown in Figure 2.

• We decided that we wanted to adapt Imperial College’s system to make our sensor less leaky by adding a second GFP linked to the C230 or LacZ enzyme. This linker will be cleaved by RecA.

• We then discussed possible issues we could have with our proposed device. First, we need to determine how to avoid a “one-time use” product by degrading the pigments after the device is used. Another aspect of the project that we need to determine is the actual material used to construct the physical device. We also need to determine the correlation between the radiation thresholds and the ribosome binding sites. The final pieces of information we need are the sequences necessary for the cleavage of the cross linkers, both for the linker cleaved by RecA and the one cleaved by tev. Then, we can construct these linkers.

Friday

• We couldn’t do a lot of lab work today because we had to order a lot of our main parts of our circuit. We were able to assemble a construct connecting a ribosome binding site to the cI repressor. Byron, Jamie, Lauren and Ben were in charge of that assembly. This assembly was done through the following steps:

1. Miniprepped C0051 (cI repressor)
2. Digest B0034 (ribosome binding site) and C0051
3. Ligated B0034 and C0051 together
4. Transform into plasmid

• Kristen and Elyse developed a presentation of our idea to Dr. Richard to welcome him back from his trip to China.

• Jim and Lauren determined and ordered the necessary primer sequence needed to remove RecA from K341452, which are given here:

Rec A Forward primer:	gagattctagATGGCTATCGACGAAAAC
Rec A Reverse primer:	tcagactgcagcggccgctactagtaTTACGACGGGATGTTGATTC

• After words of wisdom from Dr. Salis, we outlined three potential ways to “measure” DNA damage as a result of radiation exposure.

1. Change the strength of the RecA promoter
2. Change the strength of the RecA ribosome binding site
3. Change the strength of the cI ribosome binding site

• We determined that changing the strength of the ribosome binding site would most directly correlate to the radiation exposure. For example, if we use a strong cI ribosome binding site, a lot of cI repressors will be produced. The cI repressor represses our proposed reporter system. When a lot of cI repressors are present, the system needs more RecA to cleave these repressors in order for the reporter to emit pigment. In order to activate more RecA, more DNA must be damaged. To damage more DNA, the cells must be exposed to more radiation. Thus, a strong cI ribosome binding site will correlate to a large amount of radiation.

• The rest of us updated the lab notebook and continued research regarding the enzyme-GFP linkers. We also discussed the human practices project, and loosely defined groups that we will be working in to complete this project. We determined four groups: reporters, sensors, technology and RecA mutation. The reporters and sensors will probably need to be divided into smaller groups after we meet in these groups and determine their complete responsibilities.

Group Name	Group Members	Group Responsibilities
Reporters	Jim, Ben, Alex, Brian	Develop RecA- and tev-cleaved linkers connecting GFP to C230 and LacZ enzymes
Sensors	Lauren, Anisha, Jamie, Kristen	Test and compile Penn State's 2007 radiation sensor parts into one construct
RecA Mutations	Elyse, Byron, Vishal	Create mutant RecA genes that will not have recombinase qualities but still cleaves the cI repressor
Technology	All	Design Wiki page, develop human practices, update lab notebook, modelling

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