GCC Biotech Lab 3/4 - 3/8

 Introduction

    This week was a busy one in the lab. Experiments were performed in an attempt to transform Deinococcus aquaticus and give them chloramphenicol resistance. Competent D. aquaticus were made, and I learned a lot about proteins. 


Procedure
    On Monday, I learned a lot about proteins, including the basic structure of amino acids and how the form of a protein determines its function. My lab mates also performed a Gibson assembly, attempting to combine the 7kb fragment and the cmr gene. On Tuesday, we examined the plates from Friday. Unfortunately, the only plates which showed signs of growth were the positive controls. In order to check whether or not the Gibson assembly was correct, we run a gel consisting of 30mL of TAE buffer and 0.3 grams of dyed agarose. with 5uL of the 7kb fragment and 16uL of the results of yesterday's Gibson assembly. In lane one is a 1kb ladder, in lane 2 is a blank, in lane 3 are the results of the Gibson assembly, in lane 4 is a blank, in lane 5 is the 7kb fragment, in lane 6 is a blank, in lane 7 is the cmr fragment, and in lane 8 is a blank. While letting the gel sit, we prepare four TGY cultures of Deinococcus aquaticus, two flasks and two plates. The results of the gel are poor, and do not reveal the presence of the 7kb fragment combined with the ~600bp cmr gene. The following day, the plates were examined once again. No growth is visible on the chloramphenicol plates. The density of the two flasks inoculated yesterday are examined and their OD values are below 1. Thus, the flasks are not yet fit to be used. Two long-amp PCR reactions are ran in order to produce more copies of the 7kb fragment. I also learned how to use NCBI to determine of the type strain of a given bacteria have a specific gene or not. We run another gel containing the results of the PCR reaction. The results do not show the 7kb fragment being present. On Thursday, we use the nanodrop to test the concentration of the two flasks inoculated and they give OD values of 2.11 and 2.61 respectively. Gram stains were done for both flasks and they both tested gram-negative. The first flask, which had an OD value of 2.11, was diluted 20mL of TGY and the resulting concentration yielded an OD value of 1.00. The aromaticity, hydrophilicity, and percentage of sequences which code for alpha helices and beta sheets were then calculated for the CRISPR Cas 10 IIIA enzyme were then calculated using the data generated from Galaxy and Chimera. Four tubes of competent Deinococcus aquaticus are then prepared. The tubes are centrifuged at 12k rpm for one minute. The supernatant is then discarded. The pellets are resuspended with 335 uL of DI water and 1290 uL of TGy before being mixed with a mixture of 1M CaCL2 and 60% Glycerol before being stored in the -80 degree Celsius refrigerator. The following day is spent researching the protocols for the SIM, starch, oxidase, and urease characterization tests.

Results
    The results of the experiments run this week show that the Gibson assembly which was supposed to produce a transformed 7kb fragment were unsuccessful. 

Discussion
    We suspect that the reason why the transformation process failing was due to an incorrect construction of primers. This would explain both the poor gel results and the failure of the transformed E. coli to grow on the chloramphenicol plates. 

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