Genome Engineering

Design Assignment

Please answer the following questions:

1. Craig Venter has stated he will establish an IGEM style competition for the most innovative use of the JCVI minimal bacterial cell, JCVI-syn3.0. The JCVI built the organism to be used as a platform to investigate the first principles of cellular life. Rather than use the cell for a basic research question, imagine that you were head of R&D at a biotech and were trying to determine if a new biochemical pathway you had developed to fix carbon from CO2 had any industrial potential. You know the method works in cell free enzymatic reactions, but that is about all you are sure of other than the enzymes and pathway. Do not get stymied by technical details involving how you would genome engineer JCVI-syn3.0. Explain the concept and experimental rationale.

The fixation of carbondioxide to yield carbon is very significant in such a world of climate change due to greenhouse gases. There is a team that recently worked on a developing a synthetic biochemical pathway for the fixation of carbondioxide to organic carbon using an enzyme called Enoyl CoA reductase/carboxylase. The synthetic biochemical pathway with the help of this enzyme has been found to be 20 times faster than the normal biochemical pathway alias the Calvin Cycle that's been utilized by plants [1].

Similarly, if I had found a new biochemical pathway for fixation of carbon that works well on cell free enzymatic reactions, I would like to develop novel hybrid antibiotics using JCVI 3.0, by addition of genes like CCR gene, [2] that will also yield new precursors for synthesis of novel antibiotics. Also the antibiotic resistance could be

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2. In the previous classes, George Church has talked about work done in his lab to do grand scale genome engineering of E. coli to alter its genetic code. While there are similarities with how his lab engineers E. coli with how the JCVI engineers mycoplasmas, what is the fundamental difference or differences?

Differences between George Church and JCVI team

Concept	George Church Team	JCVI Team
Approach	Top down approach	Bottom up approach
Method	Modifying an existing genome using Multiplex Automated Genome Engineering(MAGE)	Designing Hypothetical Minimal Genome (HMG) using transposon mutagenesis
Composition of Genome	Contains all the genes (non essential and essential)	Contains only essential genes and quasi-essential genes
Purpose of the genes present	Altered E-coli can be used as a machinery for a wide variety of applications	It contains only genes essential for the cell to be viable and necessary for survival
Applications	Can be used for a variety of applications	Can be tailored to be used for a particular application and study the basics of the cell

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3. In the Science paper ͞Design and synthesis of a minimal bacterial genome,͟ the JCVI showed the figure below, which is the first step in an effort to rationally reorganize the minimal cell genome. As noted in the paper, when we reorganized the genome, which included separating genes in operons, as needed we placed genes behind transcriptional promoters that controlled expression of genes deleted to build minimized segment 2. We built a genome that had a reorganized segment 2, but with the other 7 segments in with their original gene order. As noted in the paper, that cell grew normally.
   

   In later experiments, the JCVI used the same strategy to design and build reorganized versions of the other 7 minimized segments and tested them as genomes that were 1/8th reorganized and 7/8ths not reorganized. None of those 7 genomes resulted in viable cells. What is your hypothesis as to why this happened?



When the 2nd segment was minimized, out of 94 genes, 53 was deleted, which might not have affected the original gene order and hence the expression of genes was undisturbed, which rendered the cell viable. When the other segments are minimized, few essential operons which might be required for transcription and translation process might be removed in order to minimize, that could have caused the cells non viable.

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4. The JCVI recently announced that it was now minimizing and reorganizing the genome of the fastest growing eukaryote, a yeast called Kluyveromyces marxianus. The goal will be to make an alternative to Saccharomyces cerevisiae that grows faster, and can be grown at higher temperature, and that would be a better platform for the kinds of biotechnology people currently use yeast for. Based on the ͞genome engineering lecture in HTGAA and the Science paper ͞Design and synthesis of a mrinimal bacterial genome͟(assigned as class reading), how would you go about minimizing and rationally reorganizing the K. marxianus? What would be different about your approach relative to how the JCVI minimized Mycoplasma mycoides to produce JCVI-syn3.0?

• Kluyveromyces marxianus is a yeast, whose genome is 10.9 mb long [3], thats about 10 times longer than Mycoplasma mycoides.
• As Kluyveromyces marxianus is an eukaryote, the minimization and reorganization of the yeast will be very complex and time consuming than that of Mycoplasma mycoides.
• As the gene structure in eukaryotes has introns as compared to the operons in prokaryotes, the minimization would be a difficult problem. The genome minimization is more prone to errors because of the complex structure, which would involve removing the introns instead of operons in prokaryotes.
• Reorganizing of the K.marxianus genome will be a herculean task as it has 5k ORFs on 8 chromosomes.
• The survival rate of the K.marxianus is very less as we are unaware of the proper functioning of all the genes present in eukaryotes.

Practical Assignment

• I grew the EPI300+pCCTIBAC cells overnight in LB Medium containing 12.5ul chloramphenicol at 37C. No growth was observed initially in the medium.


• I later inoculated 100ul of culture in LB medium only and incubated overnight. Good growth was observed.


• Later inoculated 100ul of the overnight culture grown in LB media and incubated at 37C overnight in LB media with different concentrations of chloramphenicol - 12.5ul, 10ul, 7.5ul, 5ul and 2.5ul just to check the growth of bacteria. The OD for 12.5ul chloramphenicol LB media observed with spectrophotometer at 600nm was 0.991.


• The next day 10ml of fresh LB media containing 12.5ul chloramphenicol was prepared and inoculated with 100ul of the overnight culture. The OD observed after 7-8 hours of incubation was 0.59.



• The cells were transferred into a 15ml falcon tube and then the cells were pelleted by centrifugation for 5 minutes at 6000RPM.



• The supernatant was discarded and 10ml of chilled 10% glycerol was added, then vortexed vigorously to resuspend the pellet.


• Centrifugation for 3.5 minutes at 6000RPM for washing the salt and this was repeated 5 times.



• 100ul of 10% glycerol was added to the pellet.
• The 100 ul of the culture was divided into 2 50ul aliquots in 0.5ml eppendorf tubes, sealed with parafilm and freezed until we proceed with the electroporation step.

• The cells were thawed and kept in ice.


• We have an electroporator in our laboratory but the electroporation cuvette was broken, so we followed an alternative method for transforming the E.Coli cells. The electroprator instrument available at BioNest is shown below:



• First, we kept a water bath in magnetic stirrer and heated upto 42C and a box of ice ready


• I then took out the 50ul of the electrocompetent cells (washed with glycerol) from ice and added 3ul of the helper plasmid and kept at 42C for 90 seconds, then kept at ice for 10 minutes


• After the heat shock method of transformation of E-coli cells, I took 500ul of LB media and poured on to the electrocompetant cells.


• I then put the eppendorf tube in shaker (stuck the eppendorf in shaker with a cellotape) and incubated at 37C for 1 hour.


• The cells were then spinned down for 3.5minutes at 6000 rpm and then 50ul of the cells with 30% of supernatant was plated on the LB agar containing 12.5ug/ul chloramphenicol and 20ug/ul gentamycin


• The plates were incubated at 37C overnight and waited to see if there is any growth

1st Trial - Results:
The below image shows the plate after incubation but there was no growth observed.


• We then repeated the same heat shock method of transformation again, but we tried out without washing the cells with 10% glycerol as we assumed that the electrocompetent cells were made for transformation by electroporator and we are transforming cells by heat shock method.

2nd Trial - Results:
The image below shows the plate after repeating the same protocol and we found a lot of colonies grown overnight.


• We will be confirming with a plasmid prep and running the gel to confirm that the colonies grown are the transformed bacteria or non specific bacteria.

Plasmid Prep using NucleoSpin Kit:

1. Cultivate and harvest bacterial cells
• 5ml of LB media was inoculated with biggest colonies (assuming to be our target plasmid), and allowed to grow overnight.
• Centrifuge for 30sec at 11,000g and discard the supernatant


2. Cell lysis
• 250ul of Buffer A1 was added to the pellet and vortexed
• 250ul of Buffer A2 was added and inverted the tube 6-8times (blue colour)
• Incubate at room temperature for 5 minutes
• 300ul of Buffer A3 was added and inverted 6-8 times to mix.(blue to white)


3. Clarification of lysate
• Centifuge for 5 minutes at 11,000g at room temperature


4. Bind DNA
• The supernatant is discarded
• Centrifuge for 1 minute at 11,000g


5. Wash Silica Membrane
• 500 ul of Buffer AW was preheated to 50C
• Centrifuge for 1 minute at 11,000g
• Then 600ul of Buffer A4 was added
• Centrifuge for 1 minute at 11,000g
• Discard the supernatant


6. Dry Silica Membrane
• Centrifuge for 2 minutes at 11,000g and the supernatant was discarded


7. Elute DNA
• 50ul of water was added to the pellet
• Incubate for 5 minutes at room temperature
• Centrifuge for 1 minute at 11,000g.
• The supernatant was collected from the column and the column was discarded.


• Later 1% agarose gel was casted (0.3g in 30ml 1x TAE buffer) with 2ul EtBr.


• The supernatant (plasmid that's isolated) and 1kb DNA Ladder was mixed with 2ul of DNA gel loading dye and loaded on the wells.


• The electrophoresis equipment was run for 45 minutes and then the gel was imaged under UV Transilluminator.

The band of the isolated plasmid on the gel corresponds to 12kb size, which is not the required plasmid we need for doing the transformation. As the helper plasmid has a size of about 52.7kb and the size of the TKC donor plasmid is about 8kb, the colonies that we have got is not the one, we intend to get. The transformation of E.Coli cells were not proper and we think that the colonies that were grown are non-specific.

I reconstituted the antibiotics, which were commonly available from pharmacy, as we did not want to waste time waiting for the reagents that are ordered to arrive. The chloramphenicol, supposed to be soluble in ethanol, was not dissolved completely, which might have been the reason for non specific colonies.

New ethics or safety considerations this week:

Do your activities this week raise new ethics and/or safety considerations you had not considered in week 1? Describe what activities have raised these considerations and any changes you have implemented in response.

• As far as the design experiments, there is no ethics to be considered.
• But when it comes to the practical level, the electroporator has to be used with a proper setting for E.coli, and as I am not familiar with an electroporator, got to know that the pulse should be right, else the bacteria will become non-viable.
• For the agarose gel for confirming the presence of plasmid, we used Ethidium bromide, which is carcinogenic, so I wore gloves and disposed it off safely.

REFERENCES

[1] https://phys.org/news/2016-11-synthetic-biological-metabolic-pathway-co2.html

[2] http://aem.asm.org/content/77/24/8466.full.pdf

[3] https://www.ncbi.nlm.nih.gov/pubmed/25908152