Progress Made in Engineering the 7 kb Plasmid

 

Introduction:

The 7 kb plasmid of Deinococcus aquaticus has been a long term goal for the lab to not only isolate effectively, but to genetically engineer in a way that it may potentially be useful for other Deinococcus applications in the future. The roadblocks with this plasmid began in just extraction methods from D. aquaticus, with those efforts not resulting in any significant clarity in how to most efficiently isolate this plasmid from living cells in high enough quantities to work with. Thus the idea to amplify the plasmid in a long amp procedure came to be, in which primers were made by Jonathan to not only produce considerable amounts of the plasmid but equip it with long overhangs of nucleotides that would be compatible for the Gibson assembly. So far there has been considerable difficulty validating this procedure in order to put chloramphenicol resistance (Cmr) onto the 7 kb so that this new plasmid could be tested in an array of species in the genus. This week, progress was made in the first successful visualization of the 7 kb and Gibson overhang product, and preparations are being made to attempt Gibson assembly next week. 

Methods:

Long amp PCR was used with a specific template determined the previous week, that definitively had a distinct band of the 7 kb plasmid in its entirety with an annealing temperature of 59 degrees Celsius. The results were ran on a 1% agarose gel with Gel Red, a high sensitivity dye small amounts of DNA. 

The samples from the gel were cleaned up using the Monarch DNA cleanup kit, which included a step for selectively binding to fragments over 2 kb in size to hopefully reduce the amount of contamination products that was seen in the gel. Both the 7 kb and Cmr genes were purified for optimal Gibson calculations. These samples were tested on the 1x Broad range fluorometer settings and the Nanodrop One for cleanliness. The 7 kb samples were low in concentration being about 7 ng/ul and 5 ng/ul having not been pooled, but retained high cleanliness values well over 2.00. The Cmr genes had about 40 ng/ul and 60 ng/ul respectively with also comparable cleanliness values. Gibson calculations were done using these values, as pictured below. There was another high sensitivity gel ran, but there was a mix up in dye protocols so visuals were not possible after PCR cleanup due tp low remaining concentrations. The consensus of the group was to carry out the reaction conditions labeled 1 in order to try and fit parameters of Gibson as best as we could the following week. 




Results:

 The gel pictured below is the two long amp 7 kb samples that both had smearing and two prominent bands at about 1750 and 2500 bp. It is our hope that the DNA cleanup reduced the proportion of these two major contaminants, and Gibson assembly can be successful. 


Discussion:

The next week will include an attempt at Gibson using the labeled reaction 1 calculations, and then a subsequent attempt to transform specialized E. coli cells with our newly engineered plasmid. There is still alot of questions as to whether or not our vector DNA is usable, or if the E. coli will be able to pickup and/or recognize the Deinococcus native plasmid and its ori. Another sequencing attempt will also be made at the beginning of the week, to redo the multiplexing of D. sonorensis, D. metalli, and D. xinjiangenisis given that new dilution calculations were realized and can now hopefully be implemented correctly resulting in good and usable data for future projects. 

Comments

Post a Comment

Popular posts from this blog

Investigating Production of AHL-Acylase and AHL-Lactonase in Deinococcus radiodurans

Image
 Introduction     Deinococcus radiodurans  is a gram negative extremophilic bacterial species that is frequently recognized for being able to withstand environmental conditions with high levels of radiation. It is also recently found to be one of the few, potentially only, species that produces both AHL-acylase and AHL-lactonase in order to degrade AHLs. AHL, or N-acyl homoserine lactones, are signal molecules produced by cells, typically in a biofilm community, in order to alter gene expression in the recipient cells. The communication between cells is described as quorum sensing, a survival strategy that regulates the behavior of a community of cells. Most cells that have enzymes that degrade AHLs have either AHL-acylase or AHL-lactonase, but it has been found that Deinococcus radiodurans has genes to synthesize both enzymes, which is incredibly unusual. AHL-acylase is a protein that hydrolyzes the amide bond between the lactone ring and the acyl chain of...
Read more

Evaluating Phycocyanin Extraction and Purification Methods

 Introduction:      Previous experiments have so far established that analyzing phycocyanin extraction requires two major components; approximately 1:10 dilutions of sample in appropriate buffer or deionized water, and repeated readings which can be assessed in averages. Values for purity and concentration in the past few weeks have been consistent between each other, although much lower than anticipated. The previous semester produced crude extracts that were supposedly above even food grade purity requirements,  but the authenticity of  that data is now being questioned. This week primarily focuses on comparing the exact sonication practices from the previous semester, which was primarily ultrasonic bath sonification, while also comparing data from samples centrifuged for different durations of time.  Methods:      Arturo prepared the crude extract using 40 grams of dried spirulina and 400 ml of deionized water, with the...
Read more

Evaluating Methods for Optimal Phycocyanin Extraction and Purification

 Introduction:      Attempts to purify crude extracts produced of the phycobiliprotein phycocyanin (C-PC) found in the microalgae Spirulina plantesis have been previously unsuccessful. The test results have shown that charcoal and chitosan adsorption methods for as purification purposes so far have only a negative effect on purity values, opposite to what is expected. Further testing was done in an attempt to differentiate between purification methods more in depth, beginning with primary method of purification, the charcoal adsorption column. Variations that were of interest included the use of a frit, or potentially filter paper, as well as suspending the charcoal in the crude extract as opposed to just adding charcoal to the column. It is crucial for optimal phycocyanin extraction to be able to purify crude extracts in an efficient and productive manner.  Methods:        Two tubes with one gram of spirulina and ten milliliters of deionized...
Read more