Team:BostonU/Notebook

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BostonU iGEM Team: Welcome

May

Week 5: Warming up!

We met with our mentors and got an overview of the basics of synthetic biology, namely characterization, which will be the focus of our works during the summer. We also went through the “MoClo” technique, which we intend to introduce to iGEM.

We did a survey of previous iGEM teams that got the Best Experimental Measurements award, then presented the findings to our mentors.

We performed PCR to amplify pMJSAF(LacZ) using the following MoClo forward and reverse primers: DVL0_AF with DVL0_BR (MF1), DVL0_BF with DVL0_CR (MF2), DVL0_CF with DVL0_DR (SJ1) and DVL0_DF with DVL0_ER (SJ2). We performed Gel Electrophoresis to confirm that pMJSAF was amplified in PCR: The gel ran for too long thus the bands ran off of the gel. which can be seen towards the bottom of the picture. We expect for the band to be around 500 bp.

We reperformed the PCR amplification of pMJSAF with the same pairs of primers: DVL0_AF with DVL0_BR (MF3), DVL0_BF with DVL0_CR (MF4), DVL0_CF with DVL0_DR (SJ3), DVL0_DF with DVL0_ER (SJ4). We performed Gel Electrophoresis on this set of DNA.

The amplification of pMJSAF displayed strong bands at 500 bp for the primers : DVL0_BF with DVL0_CR and DVL0_DF with DVL0_ER. However for the DVL0_AF with DVL0_BR showed a much weaker band, and the DVL0_CF with DVL0_DR did not show at all. We gel extracted MF3,4 and SJ4. Using the gel extraction protocol. We then quantified the amount of DNA we had using NanoDrop DNA quantification protocol andset up a restriction digest (SpeI, BSA, Buffer 4)for MF3,4 and SJ4. following the Restriction digest protocol

We did PCR clean up of MF3,4 and SJ4 and We quantified the product of MF3,4 and SJ4 PCR clean up using NanoDrop

For the amplification that didnìt work, we performed gradient PCR of SJ3 and MF3 in order to set the best parameters for the amplification (62-69 with interval of 1 degree C). We later found out, that our step 1 was set at 62C instead of 95C, which could have affected denaturation of the DNA template; although the SJ5-12 was amplified while the MF5-12 did not. We are unsure to the cause. The results were inconclusive compared to the Gel made previously.We ran another PCR to verify results.
We were given new primers to make stock and working solutions from. In the process we realized that we used DVL1_ER instead of DVL0_ER in SJ4, thus all SJ4 products were discarded and data obsolete. We remade the DVL1_ER stock solution by adding additional EB buffer till the right concentration has been reached. We then proceeded to make stock and working solutions for: DVL0_ER, DVL1_AF, DVL1_ER, DVL1_FF, DVL1_FR, DVL1_GF, DVL1_GR and DVL1_HR. Then we paired AF-ER, FF-GR, EF-FR, GF-HR to amplify the template DNA using PCR. We made the gel from the previously mentioned PCR of AF-ER (MS4), FF-GR (MS5), EF-FR(MS6), GF-HR (MS7) and also 3 L0 (MF3=MS1, SJ3=MS2, SJ4=MS3)

Alll the L0 failed and the L1 showed bands
We performed gel extraction on the 4 L1 primer based amplification. We performed nanodrop quantification of the products of the gel extraction.

We performed restriction digest (SpeI, BSA, Buffer 4)of the products of the gel extraction.From the restriction digest of MS4-7 we performed PCR clean up. We nano dropped the PCR clean up product.

We transformed iGEM plasmids with competent bacteriafrom stock, and plated them. We grew them overnight in 37C incubator: pSB1A3 plate 1 well 1G (1A3), pSB1AT3 plate 1 well 13A (1AT3), pSB1C3 plate 1 well 3A (1C3), pSB1K3 plate 1 well 5A (1K3). We picked colonies from the plates that were stored in the incubator and allowed the picked colonies to be incubated in a 37C shaker. We performed miniprep on 3 of the 4 overnight cultures(1k3 was not used due to minimal growth while 1A3, 1AT3 and 1C3 were able to grow sufficiently). We performed nanodrop quantification on the products of the miniprep and then performed restriction digest (SpeI, BSA, Buffer 4)of the products of the miniprep.

We made a second generation of culture of the plasmid carrying bacteria by injecting 20 uL of the original culture into 5 mL of fresh LB broth with the appropriate antibiotics. Using the generation 2 culture of the bacteria with the 1K3 plasmid, we mini prepped it. We nanodrop quantified the mini prepped material. We then ran restriction digest(SpeI, BSA, Buffer 4) of the mini prepped sample.

We streaked plates from the overnight cultures of transformed bacteria (psB1A3, pSB1AT3, pSB1C3, pSB 1k3) and placed them into the 37C incubator.
We prepared a presentation of our project for Wellesley college, whom we will be collaborating with during the summer :)
We had a meeting with professor Doug and other undergrads from CIDAR lab.