Week 1: May 16-20

From 2011.igem.org

Revision as of 19:33, 2 June 2011

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:

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

Figure 1: Proposed RecA experiment diagram

• 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

Figure 2: Radiation Sensor Diagram

• 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.

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