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Difference between revisions of "Team:CAU China/Design"

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         <p>
 
         <p>
          We contemplated ways to use synthetic biology to overcome these challenges and found that using cell extract as the central component of a manufacturing pipeline for phages might allow us to overcome these issues. With the central piece of our project in place, we were able to define the individual modules of our manufacturing pipeline. The modular approach allowed us to break down the overall problem into several isolated, solvable sub-problems that could be worked on in parallel. For each module we defined the requirements we wanted to fulfill and brainstormed potential solutions for reaching these requirements. The individual modules were designed in such a way that they are truly independent of one another and that issues in one module would not impede progress in another module. We defined quantitative criteria to measure progress in achieving the identified requirements during the build-test cycle. To maximize our chances of success and the robustness of our designs, we chose the simplest solutions we could come up with. We defined additional goals when multiple solutions fulfilling these criteria were available: accessibility, portability, affordability, and safety.    
+
        'Curiouser and curiouser!' cried Alice (she was so much surprised, that
 +
for the moment she quite forgot how to speak good English); 'now I'm
 +
opening out like the largest telescope that ever was! Good-bye, feet!'
 +
(for when she looked down at her feet, they seemed to be almost out of
 +
sight, they were getting so far off). 'Oh, my poor little feet, I wonder
 +
who will put on your shoes and stockings for you now, dears? I'm sure
 +
_I_ shan't be able! I shall be a great deal too far off to trouble
 +
myself about you: you must manage the best way you can;--but I must be
 +
kind to them,' thought Alice, 'or perhaps they won't walk the way I want
 +
to go! Let me see: I'll give them a new pair of boots every Christmas.'
 
         </p>
 
         </p>
 
         <p>
 
         <p>
   Phactory was designed to be an accessible manufacturing pipeline that produces pure, precisely defined bacteriophages at medically relevant concentrations using highly portable, affordable and modular components.         </p>
+
    
 +
Poor Alice! It was as much as she could do, lying down on one side, to
 +
look through into the garden with one eye; but to get through was more
 +
hopeless than ever: she sat down and began to cry again.     </p>
 
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           Commercially available cell extract is highly expensive, while home-made alternatives are time-consuming to produce and lack reliability. Furthermore, the presence of DNases in cell extract complicates the production of phages from linear genomes. This module therefore had two separate goals:       
+
           And so it was indeed: she was now only ten inches high, and her face
 +
brightened up at the thought that she was now the right size for going
 +
through the little door into that lovely garden. First, however, she
 +
waited for a few minutes to see if she was going to shrink any further:
 +
she felt a little nervous about this; 'for it might end, you know,' said
 +
Alice to herself, 'in my going out altogether, like a candle. I wonder
 +
what I should be like then?' And she tried to fancy what the flame of a
 +
candle is like after the candle is blown out, for she could not remember
 +
ever having seen such a thing.    
 
         </p>
 
         </p>
 
         <p>
 
         <p>
           The first goal was to produce high-quality, affordable cell extract and to establish quality control mechanisms to quantify its performance. The preparation protocols described in previous publications were analysed to identify optimization goals. <sup><a href="#phareferences">3</a></sup>  To achieve these goals every step of the preparation process was assessed and improved, with a particular focus on cell cultivation and cell lysis. To enable easy distribution and storage, we decided to improve the overall durability and storage time of our cell extract by lyophilization. After resuspension of the dried cell extract, assembly of any given bacteriophage can be performed.  
+
            
 +
After a while, finding that nothing more happened, she decided on going
 +
into the garden at once; but, alas for poor Alice! when she got to the
 +
door, she found she had forgotten the little golden key, and when she
 +
went back to the table for it, she found she could not possibly reach
 +
it: she could see it quite plainly through the glass, and she tried her
 +
best to climb up one of the legs of the table, but it was too slippery;
 +
and when she had tired herself out with trying, the poor little thing
 +
sat down and cried.
 
         </p>
 
         </p>
 
         <p>
 
         <p>
           The second goal was to optimize cell extract for the expression from linear DNA. To accomplish that, we identified the intracellular DNAse RecBCD as our main target. This enzyme - in cooperation with RecA - is an essential component maintaining the integrity of the bacterial genome<sup><a href="#phareferences">4</a></sup>. Furthermore it is the protein causing degradation of linear DNA within cell extract. Therefore, the inactivation of this endonuclease was defined as a crucial step to ensure efficient phage production within the cell extract.  
+
           'Come, there's no use in crying like that!' said Alice to herself,
 +
rather sharply; 'I advise you to leave off this minute!' She generally
 +
gave herself very good advice, (though she very seldom followed it),
 +
and sometimes she scolded herself so severely as to bring tears into
 +
her eyes; and once she remembered trying to box her own ears for having
 +
cheated herself in a game of croquet she was playing against herself,
 +
for this curious child was very fond of pretending to be two people.
 +
'But it's no use now,' thought poor Alice, 'to pretend to be two people!
 +
Why, there's hardly enough of me left to make ONE respectable person!'
 
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           Our goal was to use cell extract as a host-independent platform for phage assembly from a linear DNA template. To demonstrate the universal applicability of our manufacturing platform, we attempted to express a variety of structurally different phages at titers suitable for therapeutic application. In order to generate maximal phage titers, we identified the parameters that potentially impact phage production. These included incubation time, influence of dNTP addition and optimal DNA template concentration.
+
           Soon her eye fell on a little glass box that was lying under the table:
 +
she opened it, and found in it a very small cake, on which the words
 +
'EAT ME' were beautifully marked in currants. 'Well, I'll eat it,' said
 +
Alice, 'and if it makes me grow larger, I can reach the key; and if it
 +
makes me grow smaller, I can creep under the door; so either way I'll
 +
get into the garden, and I don't care which happens!'
 
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Revision as of 02:51, 19 June 2019










Phactory

Project Design

The Problem

Alice was beginning to get very tired of sitting by her sister on the bank, and of having nothing to do: once or twice she had peeped into the book her sister was reading, but it had no pictures or conversations in it, 'and what is the use of a book,' thought Alice 'without pictures or Presently she began again. 'I wonder if I shall fall right THROUGH the earth! How funny it'll seem to come out among the people that walk with their heads downward! The Antipathies, I think--' (she was rather glad there WAS no one listening, this time, as it didn't sound at all the right word) '--but I shall have to ask them what the name of the country is, you know. Please, Ma'am, is this New Zealand or Australia?' (and she tried to curtsey as she spoke--fancy CURTSEYING as you're falling through the air! Do you think you could manage it?) 'And what an ignorant little girl she'll think me for asking! No, it'll never do to ask: perhaps I shall see it written up somewhere.'Down, down, down. There was nothing else to do, so Alice soon began talking again. 'Dinah'll miss me very much to-night, I should think!' (Dinah was the cat.) 'I hope they'll remember her saucer of milk at tea-time. Dinah my dear! I wish you were down here with me! There are no mice in the air, I'm afraid, but you might catch a bat, and that's very like a mouse, you know. But do cats eat bats, I wonder?' And here Alice began to get rather sleepy, and went on saying to herself, in a dreamy sort of way, 'Do cats eat bats? Do cats eat bats?' and sometimes, 'Do bats eat cats?' for, you see, as she couldn't answer either question, it didn't much matter which way she put it. She felt that she was dozing off, and had just begun to dream that she was walking hand in hand with Dinah, and saying to her very earnestly, 'Now, Dinah, tell me the truth: did you ever eat a bat?' when suddenly, thump! thump! down she came upon a heap of sticks and dry leaves, and the fall was over. Alice was not a bit hurt, and she jumped up on to her feet in a moment: she looked up, but it was all dark overhead; before her was another long passage, and the White Rabbit was still in sight, hurrying down it. There was not a moment to be lost: away went Alice like the wind, and was just in time to hear it say, as it turned a corner, 'Oh my ears and whiskers, how late it's getting!' She was close behind it when she turned the corner, but the Rabbit was no longer to be seen: she found herself in a long, low hall, which was lit up by a row of lamps hanging from the roof. There were doors all round the hall, but they were all locked; and when Alice had been all the way down one side and up the other, trying every door, she walked sadly down the middle, wondering how she was ever to get out again. Suddenly she came upon a little three-legged table, all made of solid glass; there was nothing on it except a tiny golden key, and Alice's first thought was that it might belong to one of the doors of the hall; but, alas! either the locks were too large, or the key was too small, but at any rate it would not open any of them. However, on the second time round, she came upon a low curtain she had not noticed before, and behind it was a little door about fifteen inches high: she tried the little golden key in the lock, and to her great delight it fitted! Alice opened the door and found that it led into a small passage, not much larger than a rat-hole: she knelt down and looked along the passage into the loveliest garden you ever saw. How she longed to get out of that dark hall, and wander about among those beds of bright flowers and those cool fountains, but she could not even get her head through the doorway; 'and even if my head would go through,' thought poor Alice, 'it would be of very little use without my shoulders. Oh, how I wish I could shut up like a telescope! I think I could, if I only know how to begin.' For, you see, so many out-of-the-way things had happened lately, that Alice had begun to think that very few things indeed were really impossible. 1There was nothing so VERY remarkable in that; nor did Alice think it so VERY much out of the way to hear the Rabbit say to itself, 'Oh dear! Oh dear! I shall be late!' (when she thought it over afterwards, it occurred to her that she ought 2 This was the overarching problem our project needed to tackle.

Either the well was very deep, or she fell very slowly, for she had plenty of time as she went down to look about her and to wonder what was going to happen next.

fore seen a rabbit with either a waistcoat-pocket, or a watch to take out of it, and burning with curiosity, she ran across the field after it, and fortunately was just in time to see it pop down a large rabbit-hole under the hedge.

The Solution: Phactory

'Curiouser and curiouser!' cried Alice (she was so much surprised, that for the moment she quite forgot how to speak good English); 'now I'm opening out like the largest telescope that ever was! Good-bye, feet!' (for when she looked down at her feet, they seemed to be almost out of sight, they were getting so far off). 'Oh, my poor little feet, I wonder who will put on your shoes and stockings for you now, dears? I'm sure _I_ shan't be able! I shall be a great deal too far off to trouble myself about you: you must manage the best way you can;--but I must be kind to them,' thought Alice, 'or perhaps they won't walk the way I want to go! Let me see: I'll give them a new pair of boots every Christmas.'

Poor Alice! It was as much as she could do, lying down on one side, to look through into the garden with one eye; but to get through was more hopeless than ever: she sat down and began to cry again.

Preparation of Cell Extract

And so it was indeed: she was now only ten inches high, and her face brightened up at the thought that she was now the right size for going through the little door into that lovely garden. First, however, she waited for a few minutes to see if she was going to shrink any further: she felt a little nervous about this; 'for it might end, you know,' said Alice to herself, 'in my going out altogether, like a candle. I wonder what I should be like then?' And she tried to fancy what the flame of a candle is like after the candle is blown out, for she could not remember ever having seen such a thing.

After a while, finding that nothing more happened, she decided on going into the garden at once; but, alas for poor Alice! when she got to the door, she found she had forgotten the little golden key, and when she went back to the table for it, she found she could not possibly reach it: she could see it quite plainly through the glass, and she tried her best to climb up one of the legs of the table, but it was too slippery; and when she had tired herself out with trying, the poor little thing sat down and cried.

'Come, there's no use in crying like that!' said Alice to herself, rather sharply; 'I advise you to leave off this minute!' She generally gave herself very good advice, (though she very seldom followed it), and sometimes she scolded herself so severely as to bring tears into her eyes; and once she remembered trying to box her own ears for having cheated herself in a game of croquet she was playing against herself, for this curious child was very fond of pretending to be two people. 'But it's no use now,' thought poor Alice, 'to pretend to be two people! Why, there's hardly enough of me left to make ONE respectable person!'

Synthetic Phage Manufacturing

Soon her eye fell on a little glass box that was lying under the table: she opened it, and found in it a very small cake, on which the words 'EAT ME' were beautifully marked in currants. 'Well, I'll eat it,' said Alice, 'and if it makes me grow larger, I can reach the key; and if it makes me grow smaller, I can creep under the door; so either way I'll get into the garden, and I don't care which happens!'